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Abstract:

The present invention concerns compounds, compositions containing these
compounds, and methods of using these compounds and compositions as
inhibitors of Stat3 signaling, Stat3 dimerization, Stat3-DNA binding,
Stat5-DNA binding, and/or aberrant cell growth in vitro or in vivo, e.g.,
as anti-cancer agents for treatment of cancer, such as breast cancer. The
compounds of the invention include, but are not limited to, NSC 74859
(S3I-201), NSC 42067, NSC 59263, NSC 75912, NSC 11421, NSC 91529, NSC
263435, and pharmaceutically acceptable salts and analogs of the
foregoing. Other non-malignant diseases characterized by proliferation of
cells that may be treated using the compounds of the invention, but are
not limited to, cirrhosis of the liver; graft rejection; restenosis; and
disorders characterized by a proliferation of T cells such as autoimmune
diseases, e.g., type 1 diabetes, lupus and multiple sclerosis. The
invention further includes an in-vitro screening test for the presence of
malignant cells in a mammalian tissue; a method of identifying inhibitors
of constitutive Stat3 activation, Stat3-DNA binding, Stat5-DNA binding,
and/or Stat3 dimerization; and a method of identifying anti-cancer
agents.

33. A method of identifying anti-cancer agents, the method comprising
selecting a compound having a structure of Formula A-F (shown in FIGS.
10-12A-D); and determining whether the compound inhibits the growth of
cancer cells in vitro or in vivo.

[0005]Structure-based high throughput virtual screening of the National
Cancer Institute (NCI) Chemical libraries identified three compounds that
selectively inhibit Stat3 DNA-binding activity in vitro, with IC50
values of 65-86 μM, namely NSC 74859, NSC 59263, and NSC 42067. The
highest scoring compound, NSC 74859 (re-synthesized as a pure sample and
named S3I-201), selectively inhibits Stat3 DNA-binding activity in vitro
with an IC50 value of 86±33 μM. Furthermore, S3I-201 induces
growth inhibition and apoptosis of malignant cells in part by inhibiting
constitutively-active Stat3, and induces human breast tumor regression in
xenograft models. These findings support the therapeutic potential of
S3I-201 and other Stat3 inhibitors against tumors harboring aberrant
Stat3 activity.

[0006]The present invention concerns isolated compounds, compositions
containing these compounds, and methods of using these compounds and
compositions as inhibitors of Stat3 and/or as inhibitors of aberrant cell
growth, e.g., as anti-cancer agents. In one embodiment, the compound has
a structure described by Formula A, B, C, D, E, or F in FIG. 10, 11, or
12A-D, respectively, or a pharmaceutically acceptable salt or analog
thereof. In another embodiment, the compound is NSC 74859 (S3I-201; shown
in FIG. 7), NSC 59263 (shown in FIG. 8), NSC 42067 (shown in FIG. 9), NSC
75912 (shown in FIG. 50), NSC 11421 (shown in FIG. 49), NSC 91529 (shown
in FIG. 51), NSC 263435 (shown in FIG. 48), or a pharmaceutically
acceptable salt or analog of any of the foregoing. In another embodiment,
the compound is an analog of S3I-201 shown in FIGS. 13-47, i.e., a
compound selected from the group consisting of HL2-006-1, HL2-006-2,
HL2-006-3, HL2-006-4, HL2-006-5, HL2-011-1, HL2-011-2, HL2-011-3,
HL2-011-4, HL2-011-5, BG2069-1, HL2-011-6, HL2-011-7, HL2-005, HL2-003,
BG2066, BG2074, BG3004, BG3006A, BG3006B, BG3006D, BG3009, RPM381,
RPM384, RPM385, RPM405, RPM411, RPM407, RPM412, RPM408, RPM410, RPM415,
RPM416, RPM418, RPM418-A, RPM427, RPM431, RPM432, RPM444, RPM448, RPM445,
RPM447, RPM452, RPM202, or a pharmaceutically acceptable salt or analog
of any of the foregoing. In another embodiment, the compound is one
listed in Table 4, or a pharmaceutically acceptable salt or analog
thereof.

[0007]One aspect of the invention concerns a method of treating a
proliferation disorder in a subject, comprising administering an
effective amount of at least one compound of the invention to the
subject. In one embodiment, the disorder is mediated by cells harboring
constitutively-active Stat3.

[0008]Another aspect of the invention concerns a method of suppressing the
growth of malignant cells, comprising contacting the cells in vitro or in
vivo with an effective amount of at least one compound of the invention.
In one embodiment, the malignant cells harbor constitutively-active
Stat3.

[0009]Another aspect of the invention concerns a method of inducing
apoptosis in malignant cells, comprising contacting the cells in vitro or
in vivo with an effective amount of at least one compound of the
invention. In one embodiment, the malignant cells harbor
constitutively-active Stat3.

[0010]Another aspect of the invention concerns a method of inhibiting
constitutive activation of Stat3 in cells, comprising contacting the
cells in vitro or in vivo with an effective amount of at least one
compound of the invention.

[0011]Another aspect of the invention concerns a method of preventing
Stat3 dimerization in a mammalian cell, the method comprising contacting
the cell in vitro or in vivo with an effective amount of at least
compound of the invention.

[0012]Another aspect of the invention concerns a a method of disrupting
Stat3-DNA binding, the method comprising contacting the Stat3 with an
effective amount of at least one compound of the invention.

[0013]Another aspect of the invention concerns a a method of disrupting
Stat5-DNA binding, the method comprising contacting the Stat5 with an
effective amount of at least one compound of the invention.

[0014]Another aspect of the invention concerns an in-vitro screening test
for the presence of malignant cells in a mammalian tissue, the test
including: obtaining a sample containing viable cells of the tissue;
culturing the sample under conditions promoting growth of the viable
cells contained therein; treating the cultured sample with a compound of
the invention; and analyzing the treated sample by a method effective to
determine percent apoptosis of cells as an indicator of presence of
malignant cells in the sample.

[0016]Another aspect of the invention concerns a method of identifying
anti-cancer agents, the method comprising selecting a compound having a
structure of Formula A, B, C, D, E, or F (shown in FIGS. 10-12A-D); and
determining whether the compound inhibits the growth of cancer cells in
vitro or in vivo (e.g., in an animal model).

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office upon
request and payment of the necessary fee.

[0018]FIGS. 1A-1B show docking of S3I-201 (NSC 74859) to the SH2 domain of
Stat3. FIG. 1A depicts S3I-201 docked to the SH2 domain of Stat3; a
solvent accessible surface of the protein (rendered on a 6 Å shell of
residues surrounding the ligand) is shown. It is color coded according to
the electrostatic potential. Hydrogen bonding to Lys 591, Ser 611, Ser
613, and Arg 609 is shown by white dashed lines. Carbon atoms of S3I-201
are shown in green. FIG. 1B depicts S3I-201 docked to the SH2 domain of
Stat3 along with pTyr peptide. Coloring for pTyr peptide is as follows:
(P: red, Y*: pink; L: orange; K: green).

[0020]FIGS. 3A-3C show that S3I-201 suppresses Stat3-dependent but not
Stat3-independent transcriptional activity. Luciferase reporter
activities in cytosolic extracts prepared from normal mouse fibroblasts
(NIH3T3) transiently co-transfected with the Stat3-dependent (pLucTKS3
[FIG. 3A]), or the Stat3-independent (pLucSRE [FIG. 3B] or β-Casein
promoter-driven Luc [FIG. 3C]) luciferase reporters together with a
plasmid expressing the v-Src oncoprotein that activates all three
reporters, and untreated (0.05% DMSO, control) or treated with 100 μM
S3I-201 for 24. Values are the mean and S.D. of three independent
determinations.

[0021]FIGS. 4A-4G show that S3I-201 inhibits anchorage-dependent
and--independent growth only of cells that contain persistently-active
Stat3. Normal mouse fibroblasts (NIH3T3) (FIG. 4A) and v-Src transformed
counterparts (NIH3T3/v-Src) (FIG. 4B), as well as the human breast
carcinoma cells (MDA-MB-453 [FIG. 4C]), MDA-MB-435 [FIG. 4D], MDA-MB-231
[FIG. 4E], or MDA-MB-468 [FIG. 4F]) were untreated (0.05% DMSO, control)
or treated with 100 μM S3I-201 and counted by trypan blue exclusion on
each of four days for viable cell number. In FIG. 4G, v-Src transformed
mouse fibroblasts (NIH3T3/v-Src) and their v-Ras transformed counterparts
(NIH3T3/v-Ras) were grown in soft-agar suspension and untreated (0.05%
DMSO, control) or treated with 100 μM S3I-201 every 3 days until large
colonies were visible, which were enumerated. Values are the mean and
S.D. of 3-4 independent determinations.

[0022]FIGS. 5A-5C show that S3I-201 inhibits Cyclin D1, Bc1-xL and
Survivin expression and induces apoptosis in a Stat3-dependent manner. In
FIG. 5A, normal NIH3T3 mouse fibroblasts and their v-Src transformed
counterparts (NIH3T3/v-Src), and the human breast carcinoma MDA-MB-453
and MDA-MB-435 cell lines were untreated (0.05% DMSO) or treated with
100-300 μM S3I-201 for 48 hours and subjected to Annexin V staining
and Flow Cytometry. In FIG. 4B, human breast carcinoma MDA-MB-231 cells
were transfected with pcDNA3 (mock), Stat3C or untransfected (Non), or
the v-Src transformed mouse fibroblasts (NIH3T3/v-Src) were transfected
with pcDNA3 (mock), N-terminus of Stat3 (ST3-NT) or the Stat3 SH2 domain
(ST3-SH2) for 4 hours. Twenty four hours after transfection, cells were
untreated (0.05% DMSO, (-)) or treated (+) with 100 μM S3I-201 for an
additional 24 hours and subjected to Annexin V staining and Flow
Cytometry. FIG. 5C shows SDS-PAGE and Western blot analysis of whole-cell
lysates prepared from the v-Src-transformed mouse fibroblasts
(NIH3T3/v-Src) or the human breast cancer MDA-MB-231 cells untreated
(DMSO, control) or treated with 100 μM S3I-201 for 48 hours probing
with anti-Cyclin D1, Bc1-xL and Survivin antibodies. Values are the mean
and S.D. of six independent determinations. Western blot data are
representative of 3 independent analyses.

[0023]FIGS. 6A-6C show tumor growth inhibition by S3I-201. In FIG. 6A,
human breast (MDA-MB-231) tumor-bearing mice were given S3I-201 (5 mg/kg)
i.v. every 2 or every 3 days. Tumor sizes were monitored every 2 to 3
days, converted to tumor volumes, and plotted. In FIG. 6B, upon
completion of study 3 days after the last S3I-201 injection, animals were
sacrificed and tumor from one control animal (DMSO-treated) or residual
tumor tissue from S3I-201 treated (T1 and T2) mice were extracted and
lysate preparations of equal total proteins were analyzed for Stat3
activation by incubating with radiolabeled hSIE probe and subjecting to
EMSA analysis (lanes 1 to 3), or lysates from control tumor tissue of
equal protein were pre-incubated with or without increasing concentration
of S3I-201 prior to incubation with radiolabeled hSIE probe and
subjecting to EMSA analysis (lanes 1, 4, 5 and 6). In FIG. 6C, lysate
preparations from extracted tumor tissue in control or treated (T1 and
T2) were subjected to SDS-PAGE and Western blot analysis for pTyr Stat3
(pYStat3, upper panel) and total Stat3 (lower panel). Values are the mean
and S.D. of eight tumor-bearing mice each. Positions of Stat3:DNA
complexes are shown.

[0029]FIGS. 12A-12D show the structures of T-shaped molecules (Formulas C,
D, E, and F, respectively) that bind in the SH2 binding groove. The
hydrophobic group containing R2 binds in a STAT3 hydrophobic pocket
formed from Ile634, Ile597, Lys591, and Arg595. In FIGS. 12A-12D, the XNH
groups have a transposed arrangement (NHX) with respect to one another.
Referring to FIGS. 12A-12D, R1 and R2, if present, can be an
aliphatic or aromatic group; X, if present ═CO, SO2, CONH, or
alkyl; Z, if present, is alkyl; and phosphate mimic=that shown in FIGS.
10 and 11, e.g., CO2H, SO3H, PO3H, NO2,
CH2CO2H, CF2CO2H, or CF(CO2H)2 tetrazole.
Preferably, R2 is a hydrophobic group or part of a hydrophobic
group. In one embodiment, R1, if present, is H, alkyl, alkenyl,
cycloalkyl, heterocycloalkyl, cylcoalkenyl, heterocycloalkenyl, acyl, and
aryl, any of which may be optionally substituted; and R2, if present
is H, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, cylcoalkenyl,
heterocycloalkenyl, acyl, and aryl, any of which may be optionally
substituted. In a preferred embodiment, R1, if present, is aryl,
substituted aryl, heteroaryl or alkyl; and R2, if present is a
hydrophobic group such as aryl, substituted aryl, heteroaryl or alkyl.

[0030]FIG. 13 shows the chemical structure of the compound HL2-006-1, an
analog of S3I-201.

[0031]FIG. 14 shows the chemical structure of the compound HL2-006-2, an
analog of S3I-201.

[0032]FIG. 15 shows the chemical structure of the compound HL2-006-3, an
analog of S3I-201.

[0033]FIG. 16 shows the chemical structure of the compound HL2-006-4, an
analog of S3I-201.

[0034]FIG. 17 shows the chemical structure of the compound HL2-006-5, an
analog of S3I-201.

[0035]FIG. 18 shows the chemical structure of the compound HL2-011-1, an
analog of S3I-201.

[0036]FIG. 19 shows the chemical structure of the compound HL2-011-2, an
analog of S3I-201.

[0037]FIG. 20 shows the chemical structure of the compound HL2-011-3, an
analog of S3I-201.

[0038]FIG. 21 shows the chemical structure of the compound HL2-001-4, an
analog of S3I-201.

[0039]FIG. 22 shows the chemical structure of the compound HL2-011-5, an
analog of S3I-201.

[0040]FIG. 23 shows the chemical structure of the compound BG2069-1, an
analog of S3I-201.

[0041]FIG. 24 shows the chemical structure of the compound HL2-011-6, an
analog of S3I-201.

[0042]FIG. 25 shows the chemical structure of the compound HL2-011-7, an
analog of S3I-201.

[0043]FIG. 26 shows the chemical structure of the compound HL2-005, an
analog of S3I-201.

[0044]FIG. 27 shows the chemical structure of the compound HL2-003, an
analog of S3I-201.

[0045]FIG. 28 shows the chemical structure of the compound BG2066, an
analog of S3I-201.

[0046]FIG. 29 shows the chemical structure of the compound BG2074, an
analog of S3I-201.

[0047]FIG. 30 shows the chemical structure of the compound BG3004, an
analog of S3I-201.

[0048]FIG. 31 shows the chemical structure of the compound BG3006A, an
analog of S3I-201.

[0049]FIG. 32 shows the chemical structure of the compound BG3006B, an
analog of S3I-201.

[0050]FIG. 33 shows the chemical structure of the compound BG3006D, an
analog of S3I-201.

[0051]FIG. 34 shows the chemical structure of the compound BG3009, an
analog of S3I-201.

[0073]The present invention concerns isolated compounds, compositions
comprising these compounds, and methods of using these compounds and
compositions as inhibitors of Stat3 and inhibitors of aberrant cell
growth, e.g., as anti-cancer agents.

[0074]Constitutively-active Stat3 is a prevalent molecular abnormality
with a critical role in human malignant transformation, and which
represents a valid target for novel anticancer drug design. S3I-201 (NSC
74859) is a novel inhibitor of Stat3 activity identified from the
National Cancer Institute chemical libraries using structure-based
virtual screening with a computer model of the Stat3 SH2 domain bound to
its Stat3 phosphotyrosine peptide derived from the X-ray crystal
structure of the Stat3β homodimer. S3I-201 inhibits Stat3:Stat3
complex formation, and Stat3 DNA-binding and transcriptional activities.
Furthermore, S3I-201 inhibits growth and induces apoptosis preferentially
in tumors cells that contain persistently activated Stat3.
Constitutively-dimerized and active Stat3C and Stat3 SH2 domain rescue
tumor cells from S3I-201-induced apoptosis. Finally, S3I-201 inhibits the
expression of the Stat3-regulated genes Cyclin D1, Bc1-xL and Survivin,
and inhibits the growth of human breast tumors in vivo. These findings
strongly suggest that the antitumor activity of S3I-201 is mediated in
part through inhibition of aberrant Stat3 activation and provide the
proof-of-concept for the potential clinical use of Stat3 inhibitors, such
as S3I-201, in tumors harboring aberrant Stat3.

[0075]Aspects of the invention include, but are not limited to, Stat3
inhibitors, compositions comprising these compounds, and methods of using
these compounds and compositions as inhibitors of Stat3 and/or as
inhibitors of aberrant cell growth, e.g., as anti-cancer agents. In one
embodiment, the compound has a structure encompassed by Formula A, B, C,
D, E, or F in FIGS. 10-12A-D, respectively, or a pharmaceutically
acceptable salt or analog thereof. In another embodiment, the compound is
NSC 74859 (S3I-201; shown in FIG. 7), NSC 59263 (shown in FIG. 8), NSC
42067 (shown in FIG. 9), NSC 75912 (shown in FIG. 50), NSC 11421 (shown
in FIG. 49), NSC 91529 (shown in FIG. 51), NSC 263435 (shown in FIG. 48),
or a pharmaceutically acceptable salt or analog of any of the foregoing.
In another embodiment, the compound is an analog of S3I-201 shown in
FIGS. 13-47, i.e., a compound selected from the group consisting of
HL2-006-1, HL2-006-2, HL2-006-3, HL2-006-4, HL2-006-5, HL2-011-1,
HL2-011-2, HL2-011-3, HL2-011-4, HL2-011-5, BG2069-1, HL2-011-6,
HL2-011-7, HL2-005, HL2-003, BG2066, BG2074, BG3004, BG3006A, BG3006B,
BG3006D, BG3009, RPM381, RPM384, RPM385, RPM405, RPM411, RPM407, RPM412,
RPM408, RPM410, RPM415, RPM416, RPM418, RPM418-A, RPM427, RPM431, RPM432,
RPM444, RPM448, RPM445, RPM447, RPM452, and RPM202, or a pharmaceutically
acceptable salt or analog of any of the foregoing. In another embodiment,
the compound is one listed in Table 4, or a pharmaceutically acceptable
salt or analog thereof.

[0076]One aspect of the subject invention provides methods for using the
compounds of the invention as Stat3 inhibitors and/or as
anti-proliferative agents. Thus, in one embodiment, the method of the
invention comprises administering a compound of the invention to cells in
vitro or in vivo in an amount sufficient to achieve the desired result,
e.g., reduction of Stat3 activation. In another embodiment, the method
comprises administering a compound of the invention to a human or
non-human subject in an amount effective to achieve the desired
therapeutic result. In one embodiment, more than one compound of the
invention is administered to the cells in vitro or in vivo. In a
preferred embodiment, the compound is S3I-201, or a pharmaceutically
acceptable salt or analog thereof.

[0077]As used herein, the terms "treatment" and "treating", and
grammatical variations thereof, include therapy and prophylaxis. When
used as a therapy, the compounds of the invention, by themselves or in
conjunction with other agents, alleviate or reduce one or more symptoms
associated with a proliferation disorder (e.g., cancer). Thus, the
treatment methods may or may not be curative in nature. When used as a
prophylactic treatment, the compounds of the invention, by themselves or
in conjunction with other agents, delay the onset of (and may prevent)
one or more symptoms associated with a proliferation disorder (e.g.,
cancer), or may prevent the genesis of the condition.

[0078]In one aspect, the method of the invention is a method for treating
a proliferation disorder, such as cancer, comprising administering an
effective amount of a compound of the invention to a subject in need
thereof.

[0079]In another aspect, the method of the invention is a method for
inhibiting the growth of cancer cells in vitro or in vivo, comprising
administering an effective amount of a compound of the invention to the
cancer cells.

[0080]In another aspect, the subject invention provides compositions
comprising at least one isolated compound of the invention, and a
pharmaceutically acceptable carrier.

[0081]By inhibiting the growth of cells proliferating in an aberrant
manner, the methods, compounds, and compositions of the present invention
can be used to treat a number of cell proliferation disorders, such as
cancers, including, but not limited to, leukemias and lymphomas, such as
acute lymphocytic leukemia, acute non-lymphocytic leukemias, chronic
lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's Disease,
non-Hodgkin's lymphomas, and multiple myeloma, childhood solid tumors
such as brain tumors, neuroblastoma, retinoblastoma, Wilms' Tumor, bone
tumors, and soft-tissue sarcomas, common solid tumors of adults such as
lung cancer, colon and rectum cancer, breast cancer, prostate cancer,
urinary cancers, uterine cancers, bladder cancers, oral cancers,
pancreatic cancer, melanoma and other skin cancers, stomach cancer,
ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroid
cancer, esophageal cancer, and testicular cancer. The methods of the
subject invention can be carried out in vivo or in vitro, to inhibit the
growth of cells (e.g., cancer cells) in humans and non-human mammals.
Treatment for a proliferation disorder can proceed by the Stat3
inhibitor's anti-proliferative activity such as pro-apoptotic activity,
or by other mechanisms. In one embodiment, the proliferation disorder is
one on which the Stat3 inhibitor(s) act by inhibition of Stat3
DNA-binding.

[0082]Compounds of the invention having the capability to modulate (e.g.,
reduce or eliminate) signaling of the STAT3 and/or STAT5 signaling
pathway in vitro and/or in vivo, or to inhibit the growth of cancer cells
in vitro and/or in vivo by inhibition of STAT3 and/or STAT5 signaling or
a different mechanism, would be considered to have the desired biological
activity in accordance with the subject invention. For therapeutic
applications, compounds of the subject invention have the capability to
inhibit activation of the STAT3 and/or STAT5 signaling pathway, or to
inhibit the growth of cancer cells in vitro and/or in vivo by inhibition
of STAT3 and/or STAT5 signaling or a different mechanism. Inhibition of
STAT3 and/or STAT5 signaling can be assessed directly or indirectly by
various methods, including assays for inhibition of STAT3 dimerization,
inhibition of STAT3 DNA-binding, and/or inhibition of STAT5 DNA-binding,
for example. Inhibition of STAT3 and/or STAT5 signaling by compounds of
the invention selectively promotes apoptosis in tumor cells that harbor
constitutively activated STAT3. Therefore, the desirable goals of
promoting apoptosis ("programmed cell death") of selective cancerous
cells and suppression of malignant transformation of normal cells within
a patient are likewise accomplished through administration of antagonists
or inhibitors of STAT 3 signaling of the present invention, which can be
administered as simple compounds or in a pharmaceutical formulation.

[0083]In one embodiment, the proliferation disorder to be treated is a
cancer producing a tumor characterized by over-activation of Stat1,
Stat3, Stat5, or a combination of two or all three of the foregoing.
Examples of such cancer types include, but are not limited to, breast
cancer, ovarian cancer, multiple myeloma and blood malignancies, such as
acute myelogenous leukemia.

[0084]In addition to cancer, the proliferation disorder to be treated
using the compounds, compositions, and methods of the invention can be
one characterized by aberrant Stat3 activation within cells associated
with a non-malignant disease, pathological state or disorder
(collectively "disease"), and likewise comprising administering or
contacting the cells with a an effective amount of one or more Stat3
inhibitors to reduce or inhibit the proliferation. The proliferation,
hypertrophy or overgrowth of cells that is common to these diseases is
mediated by overactivation of Stat3. This protein becomes activated by a
series of biochemical events. The activation of Stat3 then leads to
another series of inter-related biochemical reactions or signal
transduction cascades that ultimately produce cell growth and division.

[0085]In one embodiment, the proliferation disorder to be treated is
characterized by a proliferation of T-cells such as autoimmune disease,
e.g., type 1 diabetes, lupus and multiple sclerosis, and pathological
states such as graft rejection induced by the presentation of a foreign
antigen such as a graft in response to a disease condition (e.g., kidney
failure). Other non-malignant diseases characterized by proliferation of
cells include cirrhosis of the liver and restenosis.

[0086]The methods of the present invention can be advantageously combined
with at least one additional treatment method, including but not limited
to, chemotherapy, radiation therapy, or any other therapy known to those
of skill in the art for the treatment and management of proliferation
disorders such as cancer.

[0087]In one embodiment, the methods and compositions of the invention
include the incorporation of a ras antagonist. Ras protein is the on/off
switch between hormone/growth factor receptors and the regulatory
cascading that result in cell division. For Ras to be activated (i.e.,
turned on) to stimulate the regulatory cascades, it must first be
attached to the inside of the cell membrane. Ras antagonist drug
development aimed at blocking the action of Ras on the regulatory
cascades has focused on interrupting the association of Ras with the cell
membrane, blocking activation of Ras or inhibiting activated Ras. The
details of the approaches to development of Ras antagonists are reviewed
in Kloog, et al., Exp. Opin. Invest. Drugs, 1999, 8(12):2121-2140. Thus,
by the term "ras antagonist", it is meant any compound or agent that
targets one or more of these phenomena so as to result in inhibition of
cell proliferation.

[0088]The Ras antagonists that may be used in conjunction with the Stat3
inhibitors of the invention affect (e.g., inhibit) the binding of Ras to
the cell membrane, which in turn reduces or inhibits the unwanted cell
proliferation. Preferred Ras antagonists include farnesyl thiosalicylic
acid (FTS) and structurally related compounds or analogs thereof, which
are believed to function by displacing or dislodging Ras from its
membrane anchor. These organic compounds may be administered parenterally
or orally. In a particularly preferred embodiment, the Ras antagonist is
formulated for oral or parenteral administration by complexation with
cyclodextrin.

[0089]While compounds of the invention can be administered to cells in
vitro and in vivo as isolated compounds, it is preferred to administer
these compounds as part of a pharmaceutical composition. The subject
invention thus further provides compositions comprising a compound of the
invention, such as those shown in FIGS. 7-9 (NSC 74859 (S3I-201), NSC
59263, NSC 42067), FIGS. 48-51 (NSC 42067, NSC 75912, NSC 11421 NSC
91529, and NSC 263435), FIGS. 10-12A-D (Formulas A, B, C, D, E, and F),
FIGS. 13-47 (HL2-006-1, HL2-006-2, HL2-006-3, HL2-006-4, HL2-006-5,
HL2-011-1, HL2-011-2, HL2-011-3, HL2-011-4, HL2-011-5, BG2069-1,
HL2-011-6, HL2-011-7, HL2-005, HL2-003, BG2066, BG2074, BG3004, BG3006A,
BG3006B, BG3006D, BG3009, RPM381, RPM384, RPM385, RPM405, RPM411, RPM407,
RPM412, RPM408, RPM410, RPM415, RPM416, RPM418, RPM418-A, RPM427, RPM431,
RPM432, RPM444, RPM448, RPM445, RPM447, RPM452, and RPM202), and listed
in Tables 4 and 5, or physiologically acceptable salt(s) or analogs of
any of the foregoing; in association with at least one pharmaceutically
acceptable carrier. The pharmaceutical composition can be adapted for
various routes of administration, such as enteral, parenteral,
intravenous, intramuscular, topical, subcutaneous, and so forth.
Administration can be continuous or at distinct intervals, as can be
determined by a person of ordinary skill in the art.

[0090]The compounds of the invention can be formulated according to known
methods for preparing pharmaceutically useful compositions. Formulations
are described in a number of sources which are well known and readily
available to those skilled in the art. For example, Remington's
Pharmaceutical Science (Martin, E. W., 1995, Easton Pa., Mack Publishing
Company, 19th ed.) describes formulations which can be used in
connection with the subject invention. Formulations suitable for
administration include, for example, aqueous sterile injection solutions,
which may contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended recipient;
and aqueous and nonaqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and may be stored in a freeze dried (lyophilized)
condition requiring only the condition of the sterile liquid carrier, for
example, water for injections, prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powder, granules,
tablets, etc. It should be understood that in addition to the ingredients
particularly mentioned above, the compositions of the subject invention
can include other agents conventional in the art having regard to the
type of formulation in question.

[0092]Pharmaceutically acceptable salts of compounds (e.g., Stat3
inhibitors) may be obtained using standard procedures well known in the
art, for example, by reacting a sufficiently basic compound such as an
amine with a suitable acid affording a physiologically acceptable anion.
Alkali metal (for example, sodium, potassium or lithium) or alkaline
earth metal (for example calcium) salts of carboxylic acids can also be
made.

[0093]As used herein, the term "analogs" refers to compounds which are
substantially the same as another compound but which may have been
modified by, for example, adding side groups, oxidation or reduction of
the parent structure. Analogs of the Stat3 inhibitors shown in FIGS. 7-9
(e.g., NSC 74859 (S3I-201), NSC 59263, and NSC 42067) FIGS. 49-52 (NSC
42067, NSC 75912, NSC 11421 NSC 91529, and NSC 263435), and other
compounds disclosed herein can be readily prepared using commonly known
standard reactions. These standard reactions include, but are not limited
to, hydrogenation, alkylation, acetylation, and acidification reactions.
Chemical modifications can be accomplished by those skilled in the art by
protecting all functional groups present in the molecule and deprotecting
them after carrying out the desired reactions using standard procedures
known in the scientific literature (Greene, T. W. and Wuts, P. G. M.
"Protective Groups in Organic Synthesis" John Wiley & Sons, Inc. New
York. 3rd Ed. pg. 819, 1999; Honda, T. et al. Bioorg. Med. Chem. Lett.,
1997, 7:1623-1628; Honda, T. et al. Bioorg. Med. Chem. Lett., 1998,
8:2711-2714; Konoike, T. et al. J Org. Chem., 1997, 62:960-966; Honda, T.
et al. J. Med. Chem., 2000, 43:4233-4246; each of which are hereby
incorporated herein by reference in their entirety). Analogs exhibiting
the desired biological activity (such as induction of apoptosis,
cytotoxicity, cytostaticity, induction of cell cycle arrest, etc.) can be
identified or confirmed using cellular assays or other in vitro or in
vivo assays. For example, assays that detect inhibition of Stat3
activation, G2/M cell cycle arrest, and/or reduction of tumor growth
may be utilized.

[0094]It will be appreciated that the compounds of the invention can
contain one or more asymmetrically substituted carbon atoms (i.e., carbon
centers). The presence of one or more of the asymmetric centers in an
analog of the invention, can give rise to stereoisomers, and in each
case, the invention is to be understood to extend to all such
stereoisomers, including enantiomers and diastereomers, and mixtures
including racemic mixtures thereof.

[0095]FIGS. 12A-12D show formulas C-F, respectively, describing compounds
of the invention. In FIGS. 12A-12D, the XNH groups have a transposed
arrangement (NHX) with respect to one another.

[0096]Referring to FIGS. 12A-12D, R1 and R2, if present, can be
an aliphatic or aromatic group; X, if present ═CO, SO2, CONH, or
alkyl; Z, if present, is alkyl; and phosphate mimic=that shown in FIGS.
10 and 11, e.g., CO2H, SO3H, PO3H, NO2,
CH2CO2H, CF2CO2H, or CF(CO2H)2 tetrazole.
Preferably, R2 is a hydrophobic group or part of a hydrophobic
group. In one embodiment, R1, if present, is H, alkyl, alkenyl,
cycloalkyl, heterocycloalkyl, cylcoalkenyl, heterocycloalkenyl, acyl, and
aryl, any of which may be optionally substituted; and R2, if present
is H, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, cylcoalkenyl,
heterocycloalkenyl, acyl, and aryl, any of which may be optionally
substituted. In a preferred embodiment, R1, if present, is aryl,
substituted aryl, heteroaryl or alkyl; and R2, if present is a
hydrophobic group such as aryl, substituted aryl, heteroaryl or alkyl.

[0097]The term "alkyl group" is intended to mean a group of atoms derived
from an alkane by the removal of one hydrogen atom. Thus, the term
includes straight or branched chain alkyl moieties including, for
example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl,
hexyl, and the like. Preferred alkyl groups contain from 1 to about 14
carbon atoms (C1-14 alkyl).

[0098]The term "aryl group" is intended to mean a group derived from an
aromatic hydrocarbon by removal of a hydrogen from the aromatic system.
Preferred aryl groups contain phenyl or substituted phenyl groups. Thus,
the term "aryl" includes an aromatic carbocyclic radical having a single
ring or two condensed rings. This term includes, for example, phenyl or
naphthyl.

[0099]The term "heteroaryl" refers to aromatic ring systems of five or
more atoms (e.g., five to ten atoms) of which at least one atom is
selected from O, N and S, and includes for example furanyl, thiophenyl,
pyridyl, indolyl, quinolyl and the like.

[0100]The term "acyl group" is intended to mean a group having the formula
RCO--, wherein R is an alkyl group or an aryl group.

[0101]The term "alkenyl" refers to a straight or branched chain alkyl
moiety having two or more carbon atoms (e.g., two to six carbon atoms,
C2-6 alkenyl) and having in addition one double bond, of either E or
Z stereochemistry where applicable. This term would include, for example,
vinyl, 1-propenyl, 1- and 2-butenyl, 2-methyl-2-propenyl, etc.

[0102]The term "cycloalkyl" refers to a saturated alicyclic moiety having
three or more carbon atoms (e.g., from three to six carbon atoms) and
which may be optionally benzofused at any available position. This term
includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
indanyl and tetrahydronaphthyl.

[0103]The term "heterocycloalkyl" refers to a saturated heterocyclic
moiety having three or more carbon atoms (e.g., from three to six carbon
atoms) and one or more heteroatom from the group N, O, S (or oxidized
versions thereof) and which may be optionally benzofused at any available
position. This term includes, for example, azetidinyl, pyrrolidinyl,
tetrahydrofuranyl, piperidinyl, indolinyl and tetrahydroquinolinyl.

[0104]The term "cycloalkenyl" refers to an alicyclic moiety having three
or more carbon atoms (e.g., from three to six carbon atoms) and having in
addition one double bond. This term includes, for example, cyclopentenyl
or cyclohexenyl.

[0105]The term "heterocycloalkenyl" refers to an alicyclic moiety having
from three to six carbon atoms and one or more heteroatoms from the group
N, O, S (or oxides thereof) and having in addition one double bond. This
term includes, for example, dihydropyranyl.

[0106]The term "halogen" means a halogen of the periodic table, such as
fluorine, chlorine, bromine, or iodine.

[0107]The term "optionally substituted" means optionally substituted with
one or more of the aforementioned groups (e.g., alkyl, aryl, heteroaryl,
acyl, alkenyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,
heterocycloalkenyl, or halogen), at any available position or positions.

[0109]The compounds of the invention are useful for various
non-therapeutic and therapeutic purposes. The compounds (e.g., Stat3
inhibitors) may be used for reducing aberrant cell growth in animals and
humans. Because of such anti-proliferative properties of the compounds,
they are useful in reducing unwanted cell growth in a wide variety of
settings including in vitro and in vivo. In addition to their use in
treatment methods, the Stat3 inhibitors of the invention are useful as
agents for investigating the role of Stat3 in cellular metabolism, and
controlling Stat3-mediated malignant or non-malignant cell growth in
vitro or in vivo. They are also useful as standards and for teaching
demonstrations.

[0110]Therapeutic application of the compounds and compositions comprising
them can be accomplished by any suitable therapeutic method and technique
presently or prospectively known to those skilled in the art. Further,
the compounds of the invention can be used as starting materials or
intermediates for the preparation of other useful compounds and
compositions.

[0111]Compounds of the invention (e.g., Stat3 inhibitors) may be locally
administered at one or more anatomical sites, such as sites of unwanted
cell growth (such as a tumor site, e.g., injected or topically applied to
the tumor), optionally in combination with a pharmaceutically acceptable
carrier such as an inert diluent. Compounds of the invention may be
systemically administered, such as intravenously or orally, optionally in
combination with a pharmaceutically acceptable carrier such as an inert
diluent, or an assimilable edible carrier for oral delivery. They may be
enclosed in hard or soft shell gelatin capsules, may be compressed into
tablets, or may be incorporated directly with the food of the patient's
diet. For oral therapeutic administration, the active compound may be
combined with one or more excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups,
wafers, aerosol sprays, and the like.

[0112]The tablets, troches, pills, capsules, and the like may also contain
the following: binders such as gum tragacanth, acacia, corn starch or
gelatin; excipients such as dicalcium phosphate; a disintegrating agent
such as corn starch, potato starch, alginic acid and the like; a
lubricant such as magnesium stearate; and a sweetening agent such as
sucrose, fructose, lactose or aspartame or a flavoring agent such as
peppermint, oil of wintergreen, or cherry flavoring may be added. When
the unit dosage form is a capsule, it may contain, in addition to
materials of the above type, a liquid carrier, such as a vegetable oil or
a polyethylene glycol. Various other materials may be present as coatings
or to otherwise modify the physical form of the solid unit dosage form.
For instance, tablets, pills, or capsules may be coated with gelatin,
wax, shellac, or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl and
propylparabens as preservatives, a dye and flavoring such as cherry or
orange flavor. Of course, any material used in preparing any unit dosage
form should be pharmaceutically acceptable and substantially non-toxic in
the amounts employed. In addition, the Stat3 inhibitor may be
incorporated into sustained-release preparations and devices.

[0113]The active agent (compounds of the invention) may also be
administered intravenously or intraperitoneally by infusion or injection.
Solutions of the active agent can be prepared in water, optionally mixed
with a nontoxic surfactant. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, triacetin, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations can
contain a preservative to prevent the growth of microorganisms.

[0114]The pharmaceutical dosage forms suitable for injection or infusion
can include sterile aqueous solutions or dispersions or sterile powders
comprising the compounds of the invention which are adapted for the
extemporaneous preparation of sterile injectable or infusible solutions
or dispersions, optionally encapsulated in liposomes. The ultimate dosage
form should be sterile, fluid and stable under the conditions of
manufacture and storage. The liquid carrier or vehicle can be a solvent
or liquid dispersion medium comprising, for example, water, ethanol, a
polyol (for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or by the use of
surfactants. Optionally, the prevention of the action of microorganisms
can be brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and
the like. In many cases, it will be preferable to include isotonic
agents, for example, sugars, buffers or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by the
inclusion of agents that delay absorption, for example, aluminum
monostearate and gelatin.

[0115]Sterile injectable solutions are prepared by incorporating the
compounds of the invention in the required amount in the appropriate
solvent with various other ingredients enumerated above, as required,
followed by filter sterilization. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods of
preparation are vacuum drying and the freeze drying techniques, which
yield a powder of the active ingredient plus any additional desired
ingredient present in the previously sterile-filtered solutions.

[0116]For topical administration, the compounds may be applied in
pure-form, i.e., when they are liquids. However, it will generally be
desirable to administer them topically to the skin as compositions, in
combination with a dermatologically acceptable carrier, which may be a
solid or a liquid.

[0117]The compounds of the subject invention can be applied topically to a
subject's skin to reduce the size (and may include complete removal) of
malignant or benign growths. The compounds of the invention can be
applied directly to the growth. Preferably, the compound is applied to
the growth in a formulation such as an ointment, cream, lotion, solution,
tincture, or the like. Drug delivery systems for delivery of
pharmacological substances to dermal lesions can also be used, such as
that described in U.S. Pat. No. 5,167,649 (Zook).

[0118]Useful solid carriers include finely divided solids such as talc,
clay, microcrystalline cellulose, silica, alumina and the like. Useful
liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the Stat3 inhibitor can be
dissolved or dispersed at effective levels, optionally with the aid of
non-toxic surfactants. Adjuvants such as fragrances and additional
antimicrobial agents can be added to optimize the properties for a given
use. The resultant liquid compositions can be applied from absorbent
pads, used to impregnate bandages and other dressings, or sprayed onto
the affected area using pump-type or aerosol sprayers, for example.

[0119]Thickeners such as synthetic polymers, fatty acids, fatty acid salts
and esters, fatty alcohols, modified celluloses or modified mineral
materials can also be employed with liquid carriers to form spreadable
pastes, gels, ointments, soaps, and the like, for application directly to
the skin of the user. Examples of useful dermatological compositions
which can be used to deliver the Stat3 inhibitors to the skin are
disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.
No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman (U.S.
Pat. No. 4,820,508).

[0120]Useful dosages of the pharmaceutical compositions of the present
invention can be determined by comparing their in vitro activity, and in
vivo activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known to the
art; for example, see U.S. Pat. No. 4,938,949.

[0121]Accordingly, the present invention includes a pharmaceutical
composition comprising a compound of the invention in combination with a
pharmaceutically acceptable carrier. Pharmaceutical compositions adapted
for oral, topical or parenteral administration, comprising an amount of a
compound of the invention constitute a preferred embodiment of the
invention. The dose administered to a patient, particularly a human, in
the context of the present invention should be sufficient to achieve a
therapeutic response in the patient over a reasonable time frame, without
lethal toxicity, and preferably causing no more than an acceptable level
of side effects or morbidity. One skilled in the art will recognize that
dosage will depend upon a variety of factors including the condition
(health) of the subject, the body weight of the subject, kind of
concurrent treatment, if any, frequency of treatment, therapeutic ratio,
as well as the severity and stage of the pathological condition.

[0122]Depending upon the disorder or disease condition to be treated, a
suitable dose(s) may be that amount that will reduce proliferation or
growth of the target cell(s). In the context of cancer, a suitable
dose(s) is that which will result in a concentration of the active agent
(the compound of the invention) in cancer tissue, such as a malignant
tumor, which is known to achieve the desired response. The preferred
dosage is the amount which results in maximum inhibition of cancer cell
growth, without unmanageable side effects. Administration of a compound
of the invention can be continuous or at distinct intervals, as can be
determined by a person of ordinary skill in the art.

[0123]To provide for the administration of such dosages for the desired
therapeutic treatment, in some embodiments, pharmaceutical compositions
of the invention can comprise between about 0.1% and 45%, and especially,
1 and 15%, by weight of the total of one or more of the compounds of the
invention based on the weight of the total composition including carrier
or diluents. Illustratively, dosage levels of the administered active
ingredients can be: intravenous, 0.01 to about 20 mg/kg; intraperitoneal,
0.01 to about 100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg;
intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about 200 mg/kg,
and preferably about 1 to 100 mg/kg; intranasal instillation, 0.01 to
about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal (body)
weight.

[0124]Mammalian species which benefit from the disclosed methods include,
but are not limited to, primates, such as apes, chimpanzees, orangutans,
humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats,
guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;
domesticated farm animals such as cows, buffalo, bison, horses, donkey,
swine, sheep, and goats; exotic animals typically found in zoos, such as
bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,
giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,
koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea
lions, elephant seals, otters, porpoises, dolphins, and whales. Other
species that may benefit from the disclosed methods include fish,
amphibians, avians, and reptiles. As used herein, the terms "patient" and
"subject" are used interchangeably and are intended to include such human
and non-human species. Likewise, in vitro methods of the present
invention can be carried out on cells of such human and non-human
species.

[0125]Patients in need of treatment using the methods of the present
invention can be identified using standard techniques known to those in
the medical or veterinary professions, as appropriate.

[0128]As used herein, the term "tumor" refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. For example, a particular
cancer may be characterized by a solid mass tumor. The solid tumor mass,
if present, may be a primary tumor mass. A primary tumor mass refers to a
growth of cancer cells in a tissue resulting from the transformation of a
normal cell of that tissue. In most cases, the primary tumor mass is
identified by the presence of a cyst, which can be found through visual
or palpation methods, or by irregularity in shape, texture or weight of
the tissue. However, some primary tumors are not palpable and can be
detected only through medical imaging techniques such as X-rays (e.g.,
mammography) or magnetic resonance imaging (MRI), or by needle
aspirations. The use of these latter techniques is more common in early
detection. Molecular and phenotypic analysis of cancer cells within a
tissue can usually be used to confirm if the cancer is endogenous to the
tissue or if the lesion is due to metastasis from another site. The
treatment methods of the invention can be utilized for early, middle, or
late stage disease, and acute or chronic disease. In some embodiments,
the tumor is characterized as one exhibiting aberrant activation of
Stat3.

[0129]According to the method of the subject invention, a compound of the
invention can be administered to a patient by itself, or co-administered
with one or more other agents such as another compound of the invention,
or a different agent or agents. Co-administration can be carried out
simultaneously (in the same or separate formulations) or consecutively.
Furthermore, according to the method of the subject invention, compounds
of the invention can be administered to a patient as adjuvant therapy.
For example, compounds can be administered to a patient in conjunction
with chemotherapy.

[0130]Thus, the compounds of the invention, whether administered
separately, or as a pharmaceutical composition, can include various other
components as additives. Examples of acceptable components or adjuncts
which can be employed in relevant circumstances include antioxidants,
free radical scavenging agents, peptides, growth factors, antibiotics,
bacteriostatic agents, immunosuppressives, anticoagulants, buffering
agents, anti-inflammatory agents, anti-angiogenics, anti-pyretics,
time-release binders, anesthetics, steroids, and corticosteroids. Such
components can provide additional therapeutic benefit, act to affect the
therapeutic action of the compounds of the invention, or act towards
preventing any potential side effects which may be posed as a result of
administration of the compounds. The Stat3 inhibitors of the subject
invention can be conjugated to a therapeutic agent, as well.

[0131]Additional agents that can be co-administered to target cells in
vitro or in vivo, such as in a patient, in the same or as a separate
formulation, include those that modify a given biological response, such
as immunomodulators. For example, proteins such as tumor necrosis factor
(TNF), interferon (such as alpha-interferon and beta-interferon), nerve
growth factor (NGF), platelet derived growth factor (PDGF), and tissue
plasminogen activator can be administered. Biological response modifiers,
such as lymphokines, interleukins (such as interleukin-1 (IL-1),
interleukin-2 (IL-2), and interleukin-6 (IL-6)), granulocyte macrophage
colony stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), or other growth factors can be administered. In one embodiment,
the methods and compositions of the invention incorporate one or more
agents selected from the group consisting of anti-cancer agents,
cytotoxic agents, chemotherapeutic agents, anti-signaling agents, and
anti-angiogenic agents.

[0137]6. The composition of any of embodiments 2-5, comprising more than
one of said compounds.

[0138]7. A method of treating a proliferation disorder in a subject,
comprising administering an effective amount of at least one compound of
embodiment 1 to the subject.

[0139]8. The method of embodiment 7, wherein said administering comprising
administering an effective amount of S3I-201 to the subject.

[0140]9. The method of embodiment 7 or 8, wherein the proliferation
disorder is cancer.

[0141]10. The method of any of embodiments 7-9, wherein the compound is
administered locally at the site of a tumor.

[0142]11. The method of any of embodiments 7-10, wherein the proliferation
disorder is cancer, and wherein the subject is suffering from a tumor and
the compound inhibits growth of the tumor.

[0143]12. The method of embodiment 7 or 8, wherein the proliferation
disorder is a non-malignant disease characterized by aberrant Stat3
activation of cells.

[0144]13. The method of any of embodiments 7-12, wherein the compound is
administered locally at the site of the proliferation disorder.

[0145]14. The method of any of embodiments 7-9, wherein the subject is not
suffering from the proliferation disorder, and wherein the compound is
administered to delay onset of the proliferation disorder.

[0146]15. The method of any of embodiments 7-14, wherein the route of
administration is selected from the group consisting of intravenous,
intramuscular, oral, and intra-nasal.

[0147]16. The method of any of embodiments 7-15, wherein the subject is
human.

[0148]17. The method of any of embodiments 7-15, wherein the subject is a
non-human mammal.

[0149]18. The method of any of embodiments 7-13 or 15-17, further
comprising identifying the subject as one suffering from the
proliferation disorder.

[0150]19. The method of embodiment 8, wherein the subject is suffering
from a tumor and wherein said administering comprises administering
S3I-201 at the site of the tumor.

[0151]20. A method of suppressing the growth of, or inducing apoptosis in,
malignant cells, the method comprising contacting the cells with an
effective amount of at least one compound of embodiment 1.

[0152]21. The method of embodiment 20, wherein said administering is
carried out in vitro.

[0153]22. The method of embodiment 20, wherein said administering is
carried out in vivo.

[0154]23. The method of any of embodiments 20-22, wherein the cells are
mammalian cancer cells.

[0155]24. The method of any of embodiments 20-23, wherein the cells
consist essentially of human breast cancer cells.

[0156]25. The method of any of embodiments 20-24, wherein said contacting
comprises contacting the cells with an effective amount of S3I-201.

[0157]26. A method of inhibiting constitutive activation of Stat3 in
cells, comprising contacting the cells with an effective amount of at
least one compound of embodiment 1.

[0158]27. The method of embodiment 26, wherein said administering is
carried out in vitro.

[0159]28. The method of embodiment 26, wherein said administering is
carried out in vivo.

[0160]29. The method of embodiment 27 or 28, wherein said contacting
comprises contacting the cells with an effective amount of S3I-201.

[0161]30. The method of any of embodiments 26-29, wherein the cells are
cancer cells.

[0162]31. The method of any of embodiments 26-30, wherein the cells
consist essentially of human breast cancer cells.

[0163]32. A method of preventing Stat3 dimerization in a mammalian cell,
the method comprising contacting the cell with an effective amount of at
least compound of embodiment 1.

[0164]33. The method of embodiment 32, wherein said contacting comprises
contacting the cell with an effective amount of S3I-201.

[0165]34. A method of disrupting Stat3-DNA binding or Stat5-DNA binding,
the method comprising contacting the Stat3 or Stat5 with an effective
amount of at least one compound of embodiment 1.

[0166]35. The method of embodiment 34, wherein said contacting comprises
contacting the Stat3 or Stat5 with an effective amount of S3I-201.

[0167]36. An in-vitro screening test for presence of malignant cells in a
mammalian tissue, the test comprising:

[0168]obtaining a sample containing viable cells of said tissue;

[0169]culturing said sample under conditions promoting growth of the
viable cells contained therein;

[0170]treating the cultured sample with a compound; and

[0171]analyzing the treated sample by a method effective to determine
percent apoptosis of cells as an indicator of presence of malignant cells
in the sample, wherein the compound is at least one compound of
embodiment 1.

[0172]37. The screening test of embodiment 36, wherein said treating
comprising contacting the cells with S3I-201.

[0174]39. The method of embodiment 38, wherein the compound is not one
selected from embodiment 1.

[0175]40. A method of identifying anti-cancer agents, the method
comprising selecting a compound having a structure of Formulas A, B, C,
D, E, or F (shown in FIGS. 10-12A-D); and determining whether the
compound inhibits the growth of cancer cells in vitro or in vivo.

[0176]41. The method of embodiment 41, wherein the compound is not one
selected from embodiment 1.

[0177]Assays known in the art and/or disclosed herein may be used to
evaluate cell apoptosis and/or inhibition of Stat signaling in carrying
out the in vitro screening test and methods set forth in the above
embodiments, such as assays for inhibition of dimerization (see, for
example, Schust, J., Berg, T., Analytical Biochemistry, 2004,
330(1):114-118, which is incorporated by reference herein in its
entirety); and assays for inhibition of DNA binding (see, for example,
Turkson J. et al., J. Biol. Chem., 2001, 276(48):45443-45455, which is
incorporated by reference herein in its entirety). Likewise, such assays
may be used to confirm the desired biological activity possessed by
analogs of the invention.

DEFINITIONS

[0178]As used herein, the terms "treat" or "treatment" refer to both
therapeutic treatment and prophylactic or preventative measures, wherein
the object is to prevent or slow down (lessen) an undesired physiological
change or disorder, such as the development or spread of cancer or other
proliferation disorder. For purposes of this invention, beneficial or
desired clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission (whether
partial or total), whether detectable or undetectable. For example,
treatment with a compound of the invention may include reduction of
undesirable cell proliferation, and/or induction of apoptosis and
cytotoxicity. "Treatment" can also mean prolonging survival as compared
to expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as well as
those prone to have the condition or disorder or those in which the
condition or disorder is to be prevented or onset delayed. Optionally,
the patient may be identified (e.g., diagnosed) as one suffering from the
disease or condition (e.g., proliferation disorder) prior to
administration of the Stat3 inhibitor of the invention.

[0179]As used herein, the term "(therapeutically) effective amount" refers
to an amount of the compound of the invention or other agent (e.g., a
drug) effective to treat a disease or disorder in a mammal. In the case
of cancer or other proliferation disorder, the therapeutically effective
amount of the agent may reduce (i.e., slow to some extent and preferably
stop) unwanted cellular proliferation; reduce the number of cancer cells;
reduce the tumor size; inhibit (i.e., slow to some extent and preferably
stop) cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit, to
some extent, tumor growth; reduce Stat3 signaling in the target cells
(such as by inhibiting the binding of DNA and Stat3), and/or relieve, to
some extent, one or more of the symptoms associated with the cancer. To
the extent the administered compound prevents growth of and/or kills
existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer
therapy, efficacy can, for example, be measured by assessing the time to
disease progression (TTP) and/or determining the response rate (RR).

[0180]As used herein, the term "growth inhibitory amount" of the compound
of the invention refers to an amount which inhibits growth or
proliferation of a target cell, such as a tumor cell, either in vitro or
in vivo, irrespective of the mechanism by which cell growth is inhibited
(e.g., by cytostatic properties, cytotoxic properties, etc.). In a
preferred embodiment, the growth inhibitory amount inhibits (i.e., slows
to some extent and preferably stops) proliferation or growth of the
target cell in vivo or in cell culture by greater than about 20%,
preferably greater than about 50%, most preferably greater than about 75%
(e.g., from about 75% to about 100%).

[0181]The terms "cell" and "cells" are used interchangeably herein and are
intended to include either a single cell or a plurality of cells, in
vitro or in vivo, unless otherwise specified.

[0182]As used herein, the term "anti-cancer agent" refers to a substance
or treatment that inhibits the function of cancer cells, inhibits their
formation, and/or causes their destruction in vitro or in vivo. Examples
include, but are not limited to, cytotoxic agents (e.g., 5-fluorouracil,
TAXOL), chemotherapeutic agents, and anti-signaling agents (e.g., the
PI3K inhibitor LY). In some embodiments, the anti-cancer agent is a ras
antagonist.

[0183]As used herein, the term "cytotoxic agent" refers to a substance
that inhibits or prevents the function of cells and/or causes destruction
of cells in vitro and/or in vivo. The term is intended to include
radioactive isotopes (e.g., At211, I131, I125, Y90,
Re186, Re188, Sm153, Bi212, P32, and radioactive
isotopes of Lu), chemotherapeutic agents, toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant or
animal origin, and antibodies, including fragments and/or variants
thereof.

[0184]As used herein, the term "chemotherapeutic agent" is a chemical
compound useful in the treatment of cancer, such as, for example,
taxanes, e.g., paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology,
Princeton, N.J.) and doxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony,
France), chlorambucil, vincristine, vinblastine, anti-estrogens including
for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and
toremifene (FARESTON, GTx, Memphis, Tenn.), and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc.
Examples of chemotherapeutic agents that may be used in conjunction with
the compounds of the invention are listed in Table 2. In a preferred
embodiment, the chemotherapeutic agent is one or more anthracyclines.
Anthracyclines are a family of chemotherapy drugs that are also
antibiotics. The anthracyclines act to prevent cell division by
disrupting the structure of the DNA and terminate its function by: (1)
intercalating into the base pairs in the DNA minor grooves; and (2)
causing free radical damage of the ribose in the DNA. The anthracyclines
are frequently used in leukemia therapy. Examples of anthracyclines
include daunorubicin (CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX),
epirubicin (ELLENCE, PHARMORUBICIN), and idarubicin (IDAMYCIN).

[0185]As used herein, the term "Stat" refers to signal transducers and
activators of transcription, which represent a family of proteins that,
when activated by protein tyrosine kinases in the cytoplasm of the cell,
migrate to the nucleus and activate gene transcription. Examples of
mammalian STATs include STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and
STAT6.

[0186]As used herein, the term "signaling" and "signaling transduction"
represents the biochemical process involving transmission of
extracellular stimuli, via cell surface receptors through a specific and
sequential series of molecules, to genes in the nucleus resulting in
specific cellular responses to the stimuli.

[0187]As used herein, the term "constitutive activation," as in the
constitutive activation of the STAT pathway, refers to a condition where
there is an abnormally elevated level of tyrosine phosphorylated STAT3
within a given cell(s), e.g., cancer cells, as compared to a
corresponding normal (e.g., non-cancer or non-transformed) cell.
Constitutive activation of STAT3 has been exhibited in a large variety of
malignancies, including, for example, breast carcinoma cell lines;
primary breast tumor specimens; ovarian cancer cell lines and tumors;
multiple myeloma tumor specimens; and blood malignancies, such as acute
myelogenous leukemia, as described in published PCT international
application WO 00/44774 (Jove, R. et al.), the disclosure of which is
incorporated herein by reference in its entirety.

[0188]Methods for determining whether a human or non-human mammalian
subject has abnormally high levels of constitutively-activated Stat3 are
known in the art and are described, for example, in U.S. patent
publication 2004-0138189-A1 and PCT publication 02/078617 A, each of
which are incorporated herein by reference in their entirety. Optionally,
the methods of the invention further comprise identifying a patient
suffering from a condition (e.g., cancer) associated with an abnormally
elevated level of tyrosine phosphorylated STAT3, or determining whether
the cancer cells can be characterized as having abnormally elevated
levels of tyrosine phosphorylated Stat3.

[0189]As used herein, the term "pharmaceutically acceptable salt or
prodrug" is intended to describe any pharmaceutically acceptable form
(such as an ester, phosphate ester, salt of an ester or a related group)
of a compound of the invention, which, upon administration to a subject,
provides the mature or base compound (e.g., a Stat3-inhibitory compound).
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids.
Suitable salts include those derived from alkali metals such as potassium
and sodium, alkaline earth metals such as calcium and magnesium, among
numerous other acids well known in the pharmaceutical art.
Pharmaceutically acceptable prodrugs refer to a compound that is
metabolized, for example hydrolyzed or oxidized, in the host to form the
compound of the present invention. Typical examples of prodrugs include
compounds that have biologically labile protecting groups on a functional
moiety of the active compound. Prodrugs include compounds that can be
oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated,
hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated,
phosphorylated, dephosphorylated to produce the active compound.

[0190]The term "pharmaceutically acceptable esters" as used herein, unless
otherwise specified, includes those esters of one or more compounds,
which are, within the scope of sound medical judgment, suitable for use
in contact with the tissues of hosts without undue toxicity, irritation,
allergic response and the like, are commensurate with a reasonable
benefit/risk ratio, and are effective for their intended use.

[0191]The terms "comprising", "consisting of" and "consisting essentially
of" are defined according to their standard meaning. The terms may be
substituted for one another throughout the instant application in order
to attach the specific meaning associated with each term.

[0192]The terms "isolated" or "biologically pure" refer to material that
is substantially or essentially free from components which normally
accompany the material as it is found in its native state.

[0193]As used in this specification, the singular forms "a", "an", and
"the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a reference to "a compound" includes more
than one such compound. A reference to "a Stat3 inhibitor" includes more
than one such inhibitor, and so forth.

[0201]Lck-SH2 domain-phosphopeptide binding assay. In vitro ELISA study
involving the Lck-SH2-GST protein and the conjugate pTyr peptide,
biotinyl-ε-Ac-EPQpYEEIEL-OH (SEQ ID NO:2) (Bachem Bioscience,
Pa.) was performed as previously described (Lee, T. R. and Lawrence, D.
S. J Med. Chem., 2000, 43:1173-1179). Briefly, 100 μl of
biotinyl-ε-Ac-EPQpYEEIEL-OH (Bachem) (in 50 mM Tris, 150 mM NaCl,
pH 7.5) was added to each well of a streptavidin-coated 96-well
microtiter plates (Pierce, Rockford, Ill.) and incubated with shaking at
4° C. overnight. Then plates were rinsed with PBS-Tween 20 and
then two times with 200 μl of BSA-T-PBS (0.2% BSA, 0.1% Tween 20,
PBS). Then 50 μl of Lck-SH2-GST (Santa Cruz Biotechnology) fusion
protein (6.4 ng/ml in BSA-T-PBS) was added to each well of the 96-well
plate in the presence and absence of 50 μl of S3I-201 (for 30 and 100
μM final concentrations) and the plate was shaken at room temperature
for 4 hours. After solutions were removed, each well was rinsed four
times with BSA-T-PBS (200 μl) and 100 μl polyclonal rabbit anti-GST
antibody (CHEMICON, Temecula, Calif.) (100 ng/ml in BSA-T-PBS) was added
to each well, incubated at 4° C. overnight. Following washing with
BSA-T-PBS, 100 μl of 200 ng/ml BSA-T-PBS horseradish peroxidase
conjugated mouse anti-rabbit antibody (Amersham Biosciences) was added to
each well and incubated for 45 minutes at room temperature. After 4
washing steps each with BSA-T-PBS and 3 washing steps each with PBS-T,
100 μl of peroxidase substrate (1-Step Turbo TMB-ELISA, Pierce) was
added to each well and incubated for 5-15 minutes. Peroxidase reaction
was stopped by adding 100 μl 1M sulfuric acid solution and absorbance
was read at 450 nm with an ELISA plate reader.

[0202]Soft-agar colony formation assay. Colony formation assays were
carried out in 6-well dishes, as described previously (Turkson, J. et al.
J. Biol. Chem., 2001, 276:45443-45455). Brie fly, each well contained 1.5
ml of 1% agarose in Dulbeco's modified Eagle's medium as the bottom layer
and 1.5 ml of 0.5% agarose in Dulbeco's modified Eagle's medium
containing 4000 or 6000 NIH3T3/v-Src or NIH3T3/v-Ras fibroblasts,
respectively, as the top layer. Treatment with S3I-201 was initiated 1
day after seeding cells by adding 100 μl of medium with or without
S3I-201, and repeating every 3 days, until large colonies were evident.
Colonies were quantified by staining with 20 μl of 1 mg/ml
iodonitrotetrazolium violet, incubating at 37° C. overnight, and
counting the next day.

[0203]Measurement of apoptosis by Flow Cytometry. Proliferating cells were
treated with or without S3I-201 for up to 48 hours. In some cases, cells
were first transfected with Stat3C, ST3-NT, or ST3-SH2 domain or
mock-transfected for 24 hours prior to treatment with compound for an
additional 24-48 hours. Cells were then detached and analyzed by Annexin
V binding (BD Biosciences, San Diego) according to the manufacturer's
protocol and Flow Cytometry to quantify the percent apoptosis.

[0204]Mice and in vivo tumor studies. Six-week-old female athymic nude
mice were purchased from Harlan (Indianapolis, Ind.) and maintained in
the institutional animal facilities approved by the American Association
for Accreditation of Laboratory Animal Care. Athymic nude mice were
injected in the left flank area s.c. with 5×106 human breast
cancer MDA-MB-231 cells in 100 μL of PBS. After 5 to 10 days, tumors
with a diameter of 3 mm were established. Animals were given S3I-201 i.v.
at 5 mg/kg every 2 or 3 days for two weeks and monitored every 2 or 3
days. Animals were stratified so that the mean tumor sizes in all
treatment were nearly identical. Tumor volume was calculated according to
the formula V=0.52×a2×b, where a, smallest superficial
diameter, b, largest superficial diameter.

[0205]It should be understood that the examples and embodiments described
herein are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in the
art and are to be included within the spirit and purview of this
application.

Example 1

Computational Modeling and Virtual Screening

[0206]The computational modeling and virtual screening study used the
GLIDE (Grid-based Ligand Docking from Energetics) software (Friesner, R.
et al. J. Med. Chem., 2004, 47:1739-1749; Halgren, T. et al. J. Med.
Chem., 2004, 47:1750-1759) (available from Schrodinger, L. L. C.) for the
docking simulations, and relied on the X-ray crystal structure of the
Stat3β homodimer bound to DNA (Becker, S. et al. Nature, 1998,
394:145-151) determined at 2.25 Å resolution (IBG1 in the Protein
Databank). The modeling approach was used as a platform for
structure-based virtual high throughput screening of the chemical
libraries of the NCI Diversity Set (≈2,400 3D structures) and the
NCI Plated Set (≈151,000 3D structures). For the virtual
screening, DNA was removed and only one of the two monomers was employed
(see FIGS. 1A and 1B). To validate the docking approach, the native pTyr
peptide, APY*LKT (SEQ ID NO: 1), was extracted from the crystal structure
of one of the monomers and docked to the other monomer, whereby GLIDE
produced a docking mode that closely resembled the X-ray crystal
structure (data not shown). Three-dimensional structures of compounds
from the NCI's chemical libraries were downloaded from the NCI DTP
website and processed with LigPrep (available from Schrodinger, L. L. C.)
to produce 2,392 3D structures for the Diversity Set and 150,829 3D
structures for the Plated Set. Then GLIDE 2.7 SP (Standard Precision
mode) docked each chemical structure (for small-molecule) into the pTyr
peptide binding site within the SH2 domain of the monomer in order to
obtain the best docking mode and docking score.

[0207]For stronger interactions of small-molecules within the Stat3 SH2
domain relative to the native pTyr peptide, emphasis is placed on
maintaining the critical atomic contacts that make the greatest
contribution to the overall binding free energy. Strong hydrogen bonding
and hydrophobic interactions within the SH2 domain are observed for
compounds identified with good docking scores, which will be predicted to
be strong binders to Stat3 and potent Stat3 inhibitors. The best score
observed from the docking studies was -11.7 kcal/mol, relative to the
native phosphopeptide sequence APY*LKT (SEQ ID NO:1) (scored as -11.9
kcal/mol). Typically, compounds receiving highly favorable scores (more
negative values) have structural features that include sulfonyl, carboxyl
and hydroxyl functional groups. For example, the current hit, S3I-201
(NSC 74859), contains all 3 groups (FIG. 7), and from the modeling data
the carboxylate moiety of S3I-201 is predicted to interact with Ser613,
Ser611, and Arg609, whereas the phenolic hydroxyl group interacts with
Lys591 of the phosphotyrosine binding site of the Stat3 SH2 domain.

Example 2

Identification of a Novel Chemical Probe as an Inhibitor of Stat3
DNA-Binding Activity

[0208]The best scoring compounds from the virtual screening studies were
selected for experimental analysis using an in vitro Stat3 DNA-binding
assay. Nuclear extracts containing activated STATs were incubated for 30
minutes with or without increasing concentrations of compounds prior to
incubation with the radiolabeled hSIE probe that binds to Stat1 and to
Stat3 or the MGFe probe that binds to Stat1 and to Stat5 and subjected to
EMSA analysis, as described under "Materials and Methods". Results for
the confirmed hit, S3I-201 (FIG. 7), show differential inhibition of
DNA-binding activities of STATs. FIG. 2A, left panel shows potent
inhibition of Stat3 DNA-binding activity by S3I-201 with an average
IC50 value of 86±33 μM (Table 3). For selectivity against STAT
family members, nuclear extract preparations from EGF-stimulated mouse
fibroblasts over-expressing the human epidermal growth factor receptor
(NIH3T3/hEGFR) containing activated Stat1, Stat3, and Stat5 were
pre-incubated with or without S3I-201 prior to incubation with the
radiolabeled probes, as described in "Materials and Methods". EMSA
analysis of the DNA-binding activities shows Stat3:Stat3 (upper),
Stat1:Stat3 (intermediate) and Stat1:Stat1 (lower) bands of complexes
with the hSIE probe (FIG. 2A-2) and Stat5:Stat5 (upper) and Stat1:Stat1
(lower) bands of complexes with the MGFe probe (FIG. 2A-2). S3I-201
preferentially inhibits Stat3 DNA-binding activity over that of Stat1,
and inhibits that of Stat5 with a 2-fold less potency (FIG. 2A-2 and
Table 3). The appearance of different degrees of activity of S3I-201 at
300 μM is due to the fact that different nuclear extract preparations
were used, one from the v-Src transformed mouse fibroblasts
(NIH3T3/v-Src) containing only activated Stat3 (FIG. 2A-1) and the other
from the EGF-stimulated NIHT3T3/hEGFR that contains activated Stat1,
Stat3, and Stat5 (FIG. 2A-2). Supershift analysis with anti-Stat3
antibody shows protein:hSIE complex (FIG. 2A-1) contains Stat3, while use
of anti-Stat1 antibody or anti-Stat5 antibody confirms protein:MGFe
complexes contain Stat1 or Stat5, respectively (FIG. 2A-2). These studies
have identified S3I-201 from the NCI chemical libraries as a potential
binder within the Stat3 SH2 domain and an inhibitor of Stat3 activation.
S3I-201 shows 2-fold preference for Stat3 over Stat5 and greater than
3-fold preference over Stat1 (FIGS. 2A-1 and 2A-2, and Table 3).

[0209]Based on the computational modeling, S3I-201 is predicted to
interact with the SH2 domain of Stat3, thereby inhibiting active Stat3
DNA-binding activity (see FIGS. 1A and 1B). To provide experimental data
in support of S3I-201's binding to Stat3, the present inventors
investigated whether unphosphorylated, inactive Stat3 monomer could
interfere with the inhibitory effect of S3I-201 on active Stat3
DNA-binding (inactive Stat3 monomer will interfere with the inhibitory
activity of S3I-201 if it interacts with the compound). To make this
determination, cell lysates of unphosphorylated, inactive Stat3 monomer
protein prepared from Sf-9 insect cells infected with only baculovirus
containing Stat3, as previously described (Turkson, J. et al. J. Biol.
Chem., 2001, 276:45443-45455; Turkson, J. et al. Mol Cancer Ther, 2004,
3:261-269; Turkson, J. et al. Mol. Cancer. Ther., 2004, 3:1533-1542;
Turkson, J. et al. J Biol. Chem., 2005, 280:32979-32988), and cell
lysates of activated Stat3 dimer protein were mixed together and the
mixture was pre-incubated with S3I-201 for 30 minutes prior to incubation
with the radiolabeled hSIE probe and EMSA analysis, as was previously
done in FIGS. 2A-1 and 2A-2. The unphosphorylated, inactive Stat3 monomer
by itself had no significant effect on DNA-binding activity of activated
Stat3 (FIG. 2B-1, compare lane 2 to lane 1), as inactive Stat3 monomer is
incapable of binding DNA (Turkson, J. et al. J Biol. Chem., 2005,
280:32979-32988). Consistent with results in FIGS. 2A-1 and 2A-2,
pre-incubation of activated Stat3 lysates with 100 μM S3I-201
completely inhibited Stat3 DNA-binding activity (FIG. 2B-1, lanes 3 and
4). By contrast, the presence of inactive Stat3 monomer diminished the
inhibitory effect of S3I-201 on the activated Stat3 in a dose-dependent
manner, resulting in the recovery of the active Stat3 DNA-binding
activity (FIG. 2B-1, lanes 5-7). The Stat3 DNA-binding activity that was
otherwise inhibited (FIG. 2B-1, lanes 3 and 4) was partially or
completely restored in the presence of 4 or 5 μl of inactive Stat3
lysates, respectively (FIG. 2B-1, lanes 6 and 7). Therefore, while the
inactive Stat3 monomer protein is unable to bind DNA, it is capable of
interacting with S3I-201 by virtue of its SH2 domain. In turn, this
interaction reduces the concentration of S3I-201 that is available to
inhibit the activated Stat3. These findings support the S3I-201:Stat3
interaction, which is consistent with the predictions from the
computational modeling, and suggest the interaction is independent of the
activation status of Stat3. To determine whether unphosphorylated,
inactive Stat1 or Stat5 monomer or the unrelated Src protein (with a SH2
domain) would have effect on S3I-201, similar studies were performed
using independently prepared cell lysates from Sf-9 insect cells infected
with only the baculovirus containing either Stat1, Stat5 or Src, as the
present inventors have previously reported (Turkson, J. et al. J. Biol.
Chem., 2001, 276:45443-45455; Turkson, J. et al. Mol Cancer Ther, 2004,
3:261-269; Turkson, J. et al. Mol. Cancer. Ther., 2004, 3:1533-1542;
Turkson, J. et al. J Biol. Chem., 2005, 280:32979-32988), and containing
either of these proteins. In contrast to the effect observed with the
inactive Stat3 monomer (FIG. 2B-1, lanes 5 to 7), EMSA analysis shows the
presence of inactive Stat1, Stat5, or Src lysate induces no significant
recovery of Stat3 DNA-binding activity (FIG. 2B-1, lanes 8 to 10, 11 to
13, and 14 to 16). In the case of the Stat1 monomer lysate, minimal
recovery of Stat3 DNA-binding activity is observed (FIG. 2B-1, lane 10),
which is evidence of a weak interaction of the Stat1 protein with
S3I-201, as revealed in the initial evaluation (FIG. 2A-2). Compared to
the effect of inactive Stat3 monomer lysates, the minimal to no effect of
inactive Stat1 or Stat5 monomer, or the unrelated Src lysate suggests
selective interaction of Stat3 with S3I-201, presumably through its SH2
domain. The amounts of proteins for each of Stat1, Stat3, or Stat5
monomer, or the Src lysate used in these studies was determined by
SDS-PAGE and Western blot analysis to be nearly similar (FIG. 2B-2, lanes
17 to 20).

[0210]To further confirm the interaction of S3I-201 with Stat3 and to
demonstrate that it blocks Stat3:Stat3 dimerization in intact cells,
Stat3 pull-down assays involving two differently-tagged Stat3 proteins,
FLAG-tagged Stat3 (FLAG-ST3) and Stat3-YFP, expressed in cells were
performed. Viral Src transformed (NIH3T3/v-Src) mouse fibroblasts stably
expressing Stat3-YFP were transiently-transfected with FLAG-ST3, and
treated either with 0.05% DMSO (control) or with S3I-201 for 24 hours and
then subjected to pull-down assay using anti-FLAG or anti-YFP antibody
and SDS-PAGE. Analysis by Western blot for FLAG of whole-cell lysates
shows equal expression of the FLAG-ST3 protein in the lysates in the
transiently-transfected cells in both the control (DMSO-treated) and
S3I-201-treated cells (FIG. 2C-2). Western blot analysis probing with
anti-FLAG antibody of the Stat3-YFP immunoprecipitates shows the presence
of FLAG-ST3 protein in the pulled-down lysate from control cells (FIG.
2C-1, left panel, upper lane 1), suggesting Stat3-YFP and FLAG-ST3
proteins were pulled down together as a complex. By contrast, Western
blot analysis probing with anti-FLAG antibody shows no detectable level
of FLAG-ST3 protein in the Stat3-YFP immunoprecipitates from
S3I-201-treated cells (FIG. 2C-1, left panel, upper lane 2 vs. lane 1),
suggesting the disruption by S3I-201 of the complex formation between
Stat3-YFP and FLAG-ST3 proteins. The amounts of Stat3-YFP in the
immunoprecipitates are shown (FIG. 2C-1, lower left panel). Similarly,
Western blot analysis probing with anti-YFP antibody of the FLAG-ST3
immunoprecipitates shows Stat3-YFP present in the pulled-down lysate from
the 0.05% DMSO-treated (control) cells (FIG. 2C-1, right panel, lower
lane 3), but significantly reduced in the FLAG-ST3 immunoprecipitates
from the S3I-201-treated cells (FIG. 2C-1, right panel, lower lane 4 vs.
lane 3). These findings suggest FLAG-ST3:Stat3-YFP complex is strongly
formed in the control cells, but is significantly diminished in the
S3I-201-treated cells. The FLAG-ST3 protein amounts in the
immunoprecipitates are shown (FIG. 2C-1, upper right panel). Together the
findings indicate that S3I-201 disrupts Stat3:Stat3 dimers, suggesting
that the compound interacts with the Stat3 SH2 domain in intact cells.

Example 4

S3I-201 does not Interfere with Lck-SH2 Domain-Phosphotyrosine Interaction

[0211]The computational modeling predicts that S3I-201 interacts with the
Stat3 SH2 domain, thereby inhibiting Stat3 DNA-binding activity (FIGS. 1A
and 1B). To further investigate the selectivity of S3I-201 and to rule
out the possibility that it interacts with other SH2 domain-containing
proteins, the present inventors evaluated its effect on the binding
between the unrelated Src family protein, Lck, and the cognate
phosphopeptide, EPQpYEEIEL (SEQ ID NO:2) (where pY represents pTyr of SEQ
ID NO:1). The present inventors used the in vitro ELISA study involving
the Lck-SH2-GST protein and the conjugate pTyr peptide,
biotinyl-ε-Ac-EPQpYEEIEL-OH (SEQ ID NO:2) (Lee, T. R. and
Lawrence, D. S. J Med. Chem., 2000, 43:1173-1179), as described in
"Materials and Methods". Results from the ELISA show that the co-presence
of the Lck-SH2-GST protein and its cognate pTyr peptide results in signal
induction (FIG. 2D, bar 4), suggesting an interaction between the two
(Lee, T. R. and Lawrence, D. S. J Med. Chem., 2000, 43:1173-1179). The
addition of 30 μM and 100 μM S3I-201 has no effect on the signal
induction (FIG. 2D, compare bars 5 and 6 to the bar 4), indicating that
S3I-201 does not interfere with the binding of the Lck SH2 domain to its
cognate pTyr peptide, EPQpYEEIEL (SEQ ID NO:2).

Example 5

S3I-201 Inhibits Stat3 Activation in Intact Cells

[0212]It has previously been shown that Stat3 is constitutively-activated
in a variety of malignant cells (Yu, C. L. et al. Science, 1995,
269:81-83; Garcia, R. et al. Oncogene, 2001, 20:2499-2513; Turkson, J. et
al. Mol. Cell. Biol., 1998, 18:2545-2552). To determine the effect of
S3I-201 on intracellular Stat3 activation, NIH3T3/v-Src mouse fibroblasts
and human breast cancer MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells that
harbor constitutively-active Stat3 were treated with the compound and
nuclear extracts prepared for Stat3 DNA-binding activity in vitro and
EMSA analysis. Compared to control (0.05% DMSO-treated cells, lane 1),
treatment with S3I-201 induced a time-dependent inhibition of
constitutive Stat3 activation in NIH3T3/v-Src fibroblasts (FIG. 2E, lanes
4-6). By 24 hours, constitutive Stat3 activation was significantly
inhibited in the v-Src-transformed mouse fibroblasts and in the human
breast cancer MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells (FIG. 2E, lanes
4-6 and 8, 10, and 12). Furthermore, SDS-PAGE and Western blot analysis
of whole-cell lysates from NIH3T3/v-Src fibroblasts show pTyr705 Stat3
levels were significantly diminished following 24-hour treatment with
S3I-201 (FIG. 2F), while total Stat3 protein level remained unchanged.
This inhibition of tyrosine phosphorylation may be explained by the fact
that by binding to the Stat3 SH2 domain, S3I-201 prevents Stat3 from
binding to the pTyr motifs of the receptor tyrosine kinases (RTKs) and
subsequently blocks de novo phosphorylation by tyrosine kinases. To
investigate non-specific effects, SDS-PAGE and Western blot analysis was
performed on whole-cell lysates from mouse fibroblasts transformed by
v-Src (NIH3T3/v-Src) or overexpressing the human EGFR (NIH3T3/hEGFR) and
stimulated by EGF to determine the ability to inhibit other signaling
proteins. Treatment with S3I-201 for 24 hours had no significant effect
on the phosphorylation of Shc (pShc), Erk1/2 (pErk1/2), or Src (pSrc) in
cells (FIGS. 2G-1 and 2G-2). Total Erk1/2 protein levels were unchanged.
Moreover, SDS-PAGE and Western blot analysis with the anti-pTyr antibody
4G10 clone shows no significant changes in the pTyr profile of
NIH3T3/v-Src fibroblasts following 24-hour treatment with S3I-201 (FIG.
2G-4), while same treatment condition significantly diminishes pTyr705
Stat3 levels (FIG. 2F). These results together indicate that at the
concentrations that inhibit Stat3 activity, S3I-201 does not
significantly interfere with other signal transduction mechanisms.

S3I-201 Blocks Anchorage-Dependent and Independent Growth Only in Cells
where Stat3 is Persistently Activated

[0214]The above results of FIGS. 2 and 3 demonstrate that S3I-201 disrupts
Stat3 activation. The present inventors next determined whether this
Stat3 activity inhibitor is able to inhibit the anchorage-dependent and
-independent (transformation) growth of human and mouse cancer cell lines
and whether this inhibition is dependent on the presence of
persistently-active Stat3. The human breast carcinoma (MDA-MB-231,
MDA-MB-435 and MDA-MB-468) cell lines and the v-Src-transformed mouse
fibroblasts (NIH3T3/v-Src) that harbor constitutively-active Stat3, and
the human breast carcinoma MDA-MB-453 cell line and normal mouse
fibroblasts (NIH3T3) that do not harbor aberrant Stat3 activity were
treated with S3I-201 and analyzed for viable cell number by trypan blue
exclusion and microscopy (FIGS. 4A-4F) or MTT assay (data not shown).
Treatment with S3I-201 significantly reduced viable cell numbers and
inhibited growth of transformed mouse fibroblasts NIH3T3/v-Src and breast
carcinoma cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) (FIGS. 4E,
4D, and 4F, respectively). By contrast, growth and viability of normal
mouse fibroblasts (NIH3T3) and breast carcinoma cell line (MDA-MB-453)
without aberrant Stat3 activity were not significantly altered (FIGS. 4A
and 4B). Thus, S3I-201 affected only those cell lines harboring aberrant
Stat3, consistent with inhibition of Stat3 DNA-binding activity (Table 3
and FIG. 2).

[0215]To further examine the effects of S3I-201 on Stat3 biological
functions, the compound was tested for its ability to inhibit the growth
of v-Src transformed mouse fibroblasts (NIH3T3/v-Src) in soft-agar
suspension in colony formation assays. Results show that growth of v-Src
transformed mouse fibroblasts in soft-agar suspension is significantly
inhibited by S3I-201 (FIG. 4G). By contrast, soft-agar growth of v-Ras
transformed counterpart (NIH3T3/v-Ras) that is independent of
constitutively-active Stat3 is unaffected by treatment with S3I-201 (FIG.
4G), indicating that S3I-201 selectively inhibits Stat3-mediated
malignant transformation.

[0216]The present inventors next determined if the S3I-201 induced loss of
tumor cell viability is due to apoptosis. To this end, the human breast
carcinoma cell lines, MDA-MB-453 and MDA-MB-435, and the normal mouse
fibroblasts (NIH3T3) and their v-Src transformed counterpart
(NIH3T3/v-Src) were untreated (0.05% DMSO, control) or treated with
S3I-201 for 48 hours and analyzed by Annexin V binding and Flow
Cytometry. At 30-100 μM, S3I-201 induced significant apoptosis in the
representative human breast carcinoma cell line, MDA-MB-435 and the
NIH3T3/v-Src, all of which harbor constitutively-active Stat3 (FIG. 5A).
The breast carcinoma MDA-MB-435 cell line is more sensitive to 30 μM
S3I-201 (FIG. 5A). By contrast, the human breast cancer MDA-MB-453 cells
and the normal mouse fibroblasts (NIH3T3) that do not contain abnormal
Stat3 activity are less sensitive to S3I-201 at 100 μM or less (FIG.
5A). These findings indicate that at concentrations that inhibit Stat3
activity, S3I-201 selectively induces apoptosis of transformed cells
harboring aberrant Stat3 signaling, suggesting the inhibition of
constitutively-active Stat3 is part of the underlying mechanism of
apoptosis by S3I-210. At 300 μM or higher, S3I-201 induced general,
non-specific cytotoxicity independent of Stat3 activation status.

[0217]The present inventors reasoned that if the ability of S3I-201 to
induce tumor cell apoptosis is due to its ability to inhibit Stat3
activation, then the constitutively-dimerized and persistently-activated
Stat3C (Bromberg, J. F. et al. Cell, 1999, 98:295-303) should rescue from
S3I-201-induced apoptosis. To this end, the breast carcinoma MDA-MB-231
cells that harbor activated Stat3 were transiently transfected with
Stat3C and evaluated for apoptosis. Twenty-four hours after transfection,
cells were treated or untreated with S3I-201 for an additional 24-48
hours, harvested and analyzed by Annexin V binding and Flow Cytometry.
Consistent with results in FIG. 5A, S3I-201 induced 50-80% apoptosis in
untransfected or mock-transfected human breast carcinoma MDA-MB-231 cells
(FIG. 5B, left panel). By contrast, cells transfected with Stat3C and
treated with S3I-201 showed greatly diminished apoptosis (FIG. 5B, left
panel)--less than 2-fold compared with 9- or 5-fold apoptosis in
non-transfected or mock-transfected cells, respectively. There is
increased in the background level of cell death, which could be due to
the effects of transfection. The rescue from the apoptotic effects of
S3I-201 can be explained on the basis that the artificially-designed
activated Stat3C is not inhibited by S3I-201 and is sufficient to promote
the biological effects of the endogenous Stat3. Thus the activated Stat3C
rescues cells from the apoptotic effects of S3I-201 by compensating for
the loss of endogenous Stat3 activity due to inhibition by S3I-201.

[0218]To establish that the effect of S3I-201 is due to its interaction
with the Stat3 SH2 domain, similar studies were performed in cells which
were transiently-transfected with either an expression vector for the
N-terminal region of Stat3 (ST3-NT) or the Stat3 SH2 domain (ST3-SH2) and
treated with or without S3I-201. Annexin V binding and Flow Cytometry
showed that while mock-transfected cells were strongly induced by S3I-201
to undergo apoptosis (FIG. 5B, left panel), similar to the
Stat3C-transfected cells, the overexpression of the Stat3-SH2 domain
diminished the apoptotic effects of S3I-201 (FIG. 5B, left panel). In
contrast, cells overexpressing the ST3-NT region showed strong induction
of apoptosis (FIG. 5B, left panel), suggesting ST3-NT has no effect on
the ability of S3I-201 to induce apoptosis of malignant cells. The
present inventors infer that the exogenous ST3-SH2 domain binds to
S3I-201, thereby preventing the compound from binding to and inhibiting
endogenous constitutively-active Stat3 protein and its biological
functions.

[0219]To investigate the molecular mechanisms for the cell growth
inhibition and apoptosis by S3I-201, the present inventors examined the
expression of the known Stat3 target genes in the v-Src-transformed mouse
fibroblasts (NIH3T3/v-Src) and the human breast carcinoma MDA-MB-231 cell
line that harbor constitutively-active Stat3. Immunoblot analysis of
whole-cell lysates shows significant reduction in expression of the
Cyclin D1, Bc1-xL and Survivin proteins in response to S3I-201 treatment
(FIG. 5C), indicating that S3I-201 represses induction of the cell cycle
and anti-apoptotic regulatory genes in malignant cells. These findings
are consistent with the biological effects induced by S3I-201 (FIGS.
3-5), and correlate with the inhibition of aberrant Stat3 activity.

Example 10

S3I-201 Induces Regression of Human Breast Tumor Xenografts

[0220]The aforementioned findings demonstrate that S3I-201 possesses a
strong inhibitory activity against aberrant Stat3, potently inhibits
anchorage-dependent and -independent tumor cell growth, and induces
apoptosis of malignant cells in a Stat3-dependent manner. The present
inventors extended these studies to evaluate the antitumor efficacy of
S3I-201 using mouse models of human breast tumor xenografts that harbor
constitutively-active Stat3. Human breast (MDA-MB-231) tumor-bearing mice
were given i.v. injection of S3I-201 or vehicle every 2 or every 3 days
for two weeks, and tumor measurements were taken every 2 to 3 days.
Compared to control (vehicle-treated) tumors, which continued to grow,
strong growth inhibition of human breast tumors were observed in mice
that received S3I-201 (FIG. 6A). Continued evaluation of treated mice
upon termination of treatment showed no resumption of tumor growth (data
not shown), suggesting potentially a long-lasting effect of S3I-201 on
tumor growth. To determine that target was inhibited by S3I-201, lysates
were prepared from tumor tissue from one control animal and from the
residual tumor tissue in two treated-mice for Stat3 DNA-binding activity
in vitro, as the present inventors have previously done (Turkson, J. et
al. Mol. Cancer. Ther., 2004, 3:1533-1542). EMSA analysis showed strong
inhibition of Stat3 DNA binding activity in residual tumor tissue from
mice treated with S3I-201 (T1 and T2) compared to control tumor (FIG. 6B,
lanes 1 to 3). SDS-PAGE and Western blot analysis of the lysates revealed
minimal to no detection of pTyr Stat3 (pYStat3) in residual tumor tissue
from treated mice (T1 and T2) compared to control (FIG. 6C, upper panel).
Total Stat3 protein remained unchanged (FIG. 6C, lower panel). Moreover,
EMSA analysis of in vitro Stat3 DNA-binding activity also shows that
pre-incubation of lysates of equal total protein from control tumor
tissue with 10, 30, and 100 μM S3I-201 prior to incubation with
radiolabeled hSIE probe resulted in a dose-dependent abrogation of Stat3
activity (FIG. 6B, lanes 4 to 6), as observed originally in FIG. 2A-1.
Together, these studies establish the proof-of-concept for the antitumor
effect of S3I-201 in tumors that harbor constitutively-active Stat3.

[0221]The data in the Examples demonstrate the feasibility of using X-ray
crystallographic data and computational modeling as a basis for a
structure-based virtual screening to identify chemical probes and
small-molecule binders of the Stat3 SH2 domain. The docking approach with
GLIDE 2.7 (Friesner, R. et al. J. Med. Chem., 2004, 47:1739-1749;
Halgren, T. et al. J. Med. Chem., 2004, 47:1750-1759) was first validated
using the native pTyr peptide, APY*LKT, which produced a docking mode
that closely resembled the X-ray crystal structure (Becker, S. et al.
Nature, 1998, 394:145-151). By this approach, the present inventors have
docked and scored small-molecules from the NCI Chemical libraries into
the Stat3 SH2 domain, and identified potent binders that ranked within
the top ˜0.1% of the docked and scored Plated Set 3D structures.
S3I-201 (NSC 74859) emerged as a potent inhibitor of Stat3 DNA-binding
activity in vitro. Similar computational analysis has been applied in the
development of inhibitors of Stat3 and other proteins (Shao, H. et al. J
Biol. Chem., 2004, 279:18967-18973; Song, H. et al. Proc Natl Acad Sci
USA, 2005, 102:4700-4705). The docking pose of S3I-201 suggests that its
aminosalicylic moiety mimics the phenylphosphate group of the PY*LKT
motif. Furthermore, its carboxyl group is hydrogen bonded to the Arg 609,
Ser 611 and Ser 613 of Stat3, while the phenolic hydroxyl group is
hydrogen-bonded to Lys 591. Moreover, the proximity of the Lys 591 to the
benzene ring that contains the phenolic hydroxyl group suggests the
possibility of a stabilizing pi-cation interaction. Also, the tolyl group
is buried within a partially hydrophobic pocket, which contains the
tetramethylene portion of the side chain of Lys 592 and the trimethylene
portion of the side chain of Arg 595, along with residues Ile 597 and Ile
634. The present inventors believe that these features (the phosphate
mimic and hydrophobic side chain) combine and explain how S3I-201 binds
to the SH2 domain of Stat3.

[0222]The native pTyr peptide inhibitor of Stat3, APY*LKT (SEQ ID NO:1),
was previously shown to disrupt Stat3:Stat3 dimerization, thereby
inducing antitumor cell effects (Turkson, J. et al. Mol Cancer Ther,
2004, 3:261-269). The computational analysis predicts that S3I-201 is a
reasonably strong binder within the Stat3 SH2 domain, raising the
possibility that it might disrupt Stat3:Stat3 dimerization as a mode of
inhibition of Stat3. Using differently-tagged Stat3 monomer proteins in
pull-down assays, the present inventors demonstrate that S3I-201 disrupts
the complex formation between two Stat3 monomers. S3I-201 preferentially
interacts with Stat3 monomer protein over Stat1 or the Src family
protein, and has a 2-fold lower potency for Stat5. In cells, the
interactions with Stat3 will prevent binding of the Stat3 SH2 to RTKs and
Src and/or Jaks and inhibit de novo phosphorylation and activation of
Stat3 monomer. Thus, S3I-201 mediates selective inhibition of Stat3
transcriptional activity, induces cell growth inhibition, loss of
viability, and apoptosis of human breast cancer and v-Src-transformed
mouse cells that harbor constitutively-active Stat3, as well as blocks
v-Src transformation, events which are all consistent with the abrogation
of Stat3 activation (Turkson, J. et al. J. Biol. Chem., 2001,
276:45443-45455; Turkson, J. et al. Mol Cancer Ther, 2004, 3:261-269;
Garcia, R. et al. Oncogene, 2001, 20:2499-2513; Catlett-Falcone, R. et
al. Immunity, 1999, 10:105-115; Mora, L. B. et al. Cancer Res, 2002,
62:6659-6666). The observation that overexpressed exogenous Stat3 SH2
domain, but not Stat3 N-terminus in malignant cells protects against
S3I-201-induced apoptosis strongly supports the Stat3 SH2 domain as the
target of S3I-201, and for that reason will bind to S3I-201 in cells,
thereby preventing the S3I-201 from inhibiting endogenous Stat3 activity.
Moreover, the rescue from the apoptotic effects of S3I-201 by the ectopic
expression of the constitutively-active Stat3C in malignant cells
demonstrates that Stat3C compensates for the absence of aberrant Stat3
signaling. Thus, these findings further strongly suggest the anti-tumor
cell effects of S3I-201 are mediated by suppressing aberrant
Stat3-induced dysregulation of the cell cycle control and the
anti-apoptotic genes. Furthermore, in vivo antitumor effects of S3I-201
in human breast tumor xenografts establish the proof-of-concept for the
therapeutic potential of S3I-201 as a Stat3 inhibitor in human tumors
that harbor constitutively-active Stat3.

[0224]The study described herein supports computational modeling
application in structure-based virtual screening for identifying Stat3
inhibitors from chemical libraries, and together with another report
(Song, H. et al. Proc Natl Acad Sci USA, 2005, 102:4700-4705) is among
the first to identify Stat3 inhibitors by this approach. S3I-201
represents a new lead for developing combinatorial libraries with
increased diversity, thereby setting a new course in the Stat3 inhibitor
design.

[0228]2-Chloro-2-oxoethyl-4-methylbenzenesulfonate (4). A mixture of
2-(tosyloxy)acetic acid (3, 400 mg, 1.74 mmol) and thionyl chloride (1
ml) was heated at reflux for 1.5 h. The excess thionyl chloride was
removed in vacuo and the resulting white solid dried under high vacuum at
50° C. for 1.5 h. The product acid chloride was used without
further purification.

[0232]General Procedure: A suspension of the aniline derivative (0.455
mmol) in water (6 ml) was stirred at room temperature for 10 min.
Na2CO3 (40 mg, 0.455 mmol) was then added and the mixture
cooled to 0° C. A solution of
2-chloro-2-oxoethyl-4-methylbenzenesulfonate (4, 125 mg, 0.5 mmol) in THF
(2 ml) was injected rapidly and the resulting solution allowed to warm to
room temperature and stirred for 2 h.

[0238]2-(4-Methylphenylsulfonamido)acetyl chloride (10). A mixture of
2-(4-methylphenylsulfonamido)acetic acid (9, 200 mg, 0.873 mmol) and
thionyl chloride (1 ml) was heated at reflux for 1.5 h. The excess
thionyl chloride was removed in vacuo and the resulting white solid dried
under high vacuum at 50° C. for 1.5 h. The product acid chloride
10 was used without further purification.

[0242]2-Chloro-2-oxoethyl 4'-chlorobiphenyl-4-sulfonate (14). A mixture of
acid 13 (100 mg, 0.306 mmol) and thionyl chloride (1 ml) was heated at
reflux for 1.5 h. The excess thionyl chloride was removed in vacuo and
the resulting pale yellow/white solid dried under high vacuum at
50° C. for 1.5 h. The product acid chloride 14 was used without
further purification.

[0243]4-(2-(4'-Chlorobiphenyl-4-ylsulfonyloxy)acetamido)-2-hydroxybenzoic
acid (15). A mixture of p-aminosalicylic acid (43 mg, 0.278 mmol) and
NaOH (11 mg, 0.278 mmol) in water (6 ml) was stirred at room temperature
for 10 min until all of the solid material had dissolved.
Na2CO3 (25 mg, 0.231 mmol) was then added and the mixture
cooled to 0° C. A suspension of 14 (105 mg, 0.306 mmol) in THF (2
ml) was injected rapidly and the resulting solution allowed to warm to
room temperature and stirred for 2 h. Then reaction mixture was poured
into a separating funnel containing diethyl ether. The phases were
separated and the aqueous phase washed with a further portion of diethyl
ether. The aqueous phase was acidified to pH 1 by addition of 1N HCl
solution and the product extracted into ethyl acetate. The ethyl acetate
was washed twice with 1N HCl solution and the organic phase was dried
(Na2SO4) and filtered. The product was found to be in both the
diethyl ether and ethyl acetate fractions so these organic phases were
combined and the solvent removed in vacuo. The product was purified by
column chromatography over silica gel (Rf=0.27, 10% MeOH in ethyl
acetate) affording 15 as a yellow powder (45 mg, 35%). An analytical
sample was obtained by further purification by recrystallization from
EtOAc/MeOH. δH (400 MHz, MeOD) 4.72 (2H, s, CH2), 6.89
(1H, dd, J=8.6 Hz and 1.8 Hz, ArH), 7.13 (1H, d, J=1.8 Hz, ArH), 7.46
(2H, d, J=8.4 Hz, ArH), 7.58 (2H, d, J=8.4 Hz, ArH), 7.70 (1H, d, J=8.6
Hz, ArH), 7.82 (2H, d, J=8.4 Hz, ArH), 8.03 (2H, d, J=8.4 Hz, ArH); MS
(ES-) 401.9 (100%), 459.8 (75%, [M-H]-).

[0246]2-Chloro-2-oxoethyl biphenyl-4-sulfonate (18). A mixture of acid 17
(200 mg, 0.685 mmol) and thionyl chloride (1 ml) was heated at reflux for
1.5 h. The excess thionyl chloride was removed in vacuo and the residue
dried under high vacuum at 50° C. for 1.5 h. The product acid
chloride 18 was used without further purification.

[0247]4-(2-(biphenyl-4-ylsulfonamido)acetamido)-2-hydroxybenzoic acid
(19). A mixture of p-aminosalicylic acid (95 mg, 0.623 mmol) and NaOH (25
mg, 0.623 mmol) in water (6 ml) was stirred at room temperature for 10
min until all of the solid material had dissolved. Na2CO3 (55
mg, 0.517 mmol) was then added and the mixture cooled to 0° C. A
suspension of 18 (213 mg, 0.685 mmol) in THF (2 ml) was injected rapidly
and the resulting solution allowed to warm to room temperature and
stirred for 2 h. Then reaction mixture was poured into a separating
funnel containing diethyl ether. The phases were separated and the
aqueous phase washed with a further portion of diethyl ether. The aqueous
phase was acidified to pH 1 by addition of 1N HCl solution and the
product extracted into ethyl acetate. The ethyl acetate was washed twice
with 1N HCl solution and the organic phase was dried (Na2SO4),
filtered and the solvent removed in vacuo. The product was found to be in
both the diethyl ether and ethyl acetate fractions so these organic
phases were combined and the solvent removed in vacuo. The product was
purified by column chromatography over silica gel (Rf=0.22, 10% MeOH
in ethyl acetate) affording 19 as a yellow powder (109 mg, 41%). An
analytical sample was obtained by further purification by
recrystallization from EtOAc/hexane. δH (400 MHz, DMSO) 4.76
(2H, s, CH2), 6.96 (1H, d, J=8.4 Hz, ArH), 7.22 (1H, s, ArH),
7.42-7.74 (6H, m, ArH), 7.93-8.01 (4H, m, ArH), 10.37 (1H, s, NH); MS
(ES.sup.+) 428.0 (100%, [M+H].sup.+).

[0249]The following compounds were prepared according to similar method as
shown in Scheme 3

##STR00004##

32 and 31 were synthesized as outlined in the Scheme 3

[0250]Synthesis of the intermediate 1a: The sulfonyl chloride (0.995 g,
3.94 mmol) was suspended in pyridine:DCM (10 ml: 5 ml) and ethyl
glycinate (0.500 g, 3.58 mmol) was added at room temperature under argon
atmosphere, and stirred at room temperature for 4 h. The reaction mixture
was diluted with EtOAc and washed with 4M HCl followed by brine. The
organic phase was dried (MgSO4) and concentrated to obtain the
intermediate 1a (0.941 g). This intermediate was carried to the next
stage without purification.

[0251]The intermediate 1b was synthesized using the same procedure.

[0252]Synthesis of the intermediate 2a: The intermediate 1a (900 mg, 2.82
mmol) was suspended in EtOH:H2O (9 ml: 1 ml) and NaOH (5% wt, 50 mg)
was added to the reaction mixture and refluxed for 3 h. The reaction
mixture was cooled to room temperature, acidified (pH=2-3) and extracted
with EtOAc. The combined organic phase was dried (MgSO4) and
concentrated to obtain the required acid (770 mg) as a white powder. The
carboxylic acid obtained was refluxed with thionyl chloride:DCM (3:1) for
2-3 h. The excess thionyl chloride was removed under vacuum to give the
intermediate 2a (820 mg). This product was carried to the next stage.

[0271]4-Phenoxybenzenesulfonyl chloride. A solution of
4-phenoxybenzenesulfonic acid (3.14 g, 12.64 mmol) in thionyl chloride (9
ml) was refluxed for 4 h in presence of DMF (2 drops). After cooling to
room temperature, diethyl ether (20 ml) was added and the resulting white
solid was separated by filtration. The filtered solution was evaporated
in vacuo to give 4-phenoxybenzenesulfonyl chloride as a brown oil which
was used in the next step without further purification.

[0279]Methyl 4-(3-phenylacryloylamino)benzoate (41). This was obtained as
a white solid from cinnamoyl chloride (1.1 g, 6.62 mmol) and ethyl
4-aminobenzoate (1.10 g, 6.62 mmol) in a similar manner as described for
preparation of 42. The reaction mixture was stirred at room temperature
overnight and the mixture was poured in HCl (aq, 2N, 20 ml). The product
was extracted with DCM (2×20 ml), dried over Na2SO4 and
the solvent removed under reduced pressure. The crude amide 41 was used
in the next step without further purification.

[0286]Ethyl 4-(4-amino-3-phenylbutanamido)benzoate (51). Ref. J. Org.
Chem., 2000, 65, 8001. To a stirred solution of nitro compound 47 (0.749
g, 2.10 mmol) and NiCl2.6H2O (1.99 g, 8.40 mmol) in methanol
(10 ml) NaBH4 (0.719 g, 18.93 mmol) was added portionwise over 20
min at 0° C. After stirring for 15 at room temperature, the
solvent was removed under reduced pressure. Water (20 ml) and ethyl
acetate (40 ml) were added to the solid residue. The resulting mixture
was filtered through a celite bed which was washed with ethyl acetate (20
ml). After collecting the filtrate, the organic phase was separated,
dried over Na2SO4 and the solvent removed under reduced
pressure to afford amine 51 as an off white solid (0.596 g, 1.83 mmol,
87%). The amine was used in the next step without further purification.

[0287]Ethyl 4-(4-amino-3-(naphthalen-2-yl)butanamido)benzoate (52). This
was obtained as a yellow solid (0.819 g, 2.178 mmol, 89%) from nitro
compound 48 (0.995 g, 2.450 mmol) in a similar manner as described for
preparation of 51. The product amine 52 used in the next step without
further purification.

[0288]Methyl
4-(4-amino-3-(naphthalen-2-yl)butanamido)-2-(benzyloxy)benzoate (53).
This was obtained as a white solid (0.023 g, 0.033 mmol, 36%) from nitro
compound 49 (0.043 g, 0.091 mmol) a similar manner as described for
preparation of 51. The product amine 53 was taken to the next step
without further purification.

[0289]Methyl
4-(4-amino-3-(4-chlorophenyl)butanamido)-2-(benzyloxy)benzoate (54). This
was obtained as yellow solid (0.349 g, 0.79 mmol, 77%) from nitro
compound 50 (0.488 g, 1.033 mmol) a similar manner as described for
preparation of 51. The crude amine 54 was taken to the next step without
further purification.

[0298]Methyl
2-(benzyloxy)-4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butana-
mido)benzoate (63). This was obtained as an off white solid (0.023 g,
0.032 mmol, 36%) from amine 53 (0.043 g, 0.091 mmol) in a similar manner
as described for preparation of 55. The crude ester was used in the next
step without further purification.

[0299]Methyl
2-hydroxy-4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butanamido-
)benzoate (64). A solution of 63 (0.023 g, 0.032 mmol) in methanol (1 ml)
was stirred in presence of ammonium formate (0.089 g) and 10% Pd/C (0.023
g), under H2, at room temperature for 2 days. The solution was then
filtered through a celite bed and the solvent removed under reduced
pressure to afford ester 64 (0.014 g, 0.0230 mmol, 70%) as yellow solid,
which was used in the next step without further purification.

[0301]Methyl
2-(benzyloxy)-4-(3-(4-chlorophenyl)-4-(4-phenoxyphenylsulfonamido)butanam-
ido)benzoate (66). This was obtained as an off-white solid (0.205 g, 0.304
mmol, 78%) from the amine 54 (0.137 g, 0.391 mmol) in a similar manner as
described for preparation of 55. The crude product sulfonamide was taken
to the next step without further purification.

[0302]Methyl
4-(3-(4-chlorophenyl)-4-(4-phenoxyphenylsulfonamido)butanamido)-2-hydroxy-
benzoate (67). This was obtained as an off-white solid (0.106 g, 0.181
mmol, 67%) from benzyl ether 66 (0.183 g, 0.27 mmol) in a similar manner
as described for preparation of 64. The crude ester 67 was taken to the
next step without further purification.

[0312]The following experiments and results are described and shown in
Siddiquee, K. et al., PNAS USA, 2007, May, 104(18):7391-7396, Epub 2007
Apr. 26, which is incorporated by reference herein in its entirety.
Nuclear extracts containing activated Stat1, Stat3, and Stat5 proteins
were pre-incubated with increasing concentrations of compounds for 30
minutes at room temperature prior to incubation with radiolabeled hSIE
probe that binds Stat1 and Stat3 or MGFe probe that binds Stat1 and Stat5
and subjecting to EMSA analysis. Cell lysates containing activated Stat3
were pre-incubated with NSC 74859 in the presence and absence of
increasing amount of cell lysates containing inactive Stat3 protein
(monomer) prior to incubation with radiolabeled hSIE probe and subjecting
to EMSA analysis. Nuclear extract preparations from v-Src-transformed
NIH3T3/v-Src fibroblasts treated for the times indicated in FIG. 2 of
Siddiquee, K et al. (2007) or human breast cancer MDA-MB-231 and
MDA-MB-468 treated for 48 hours with 100 μM NSC 74859 were incubated
with radiolabeled hSIE probe and subjected to EMSA analysis. SDS-PAGE and
Western blot analysis of whole-cell lysates from NIH3T3/v-Src fibroblasts
treated with or without NSC 74859 (100 μM, 48 hours) for pTyr705Stat3
or Stat3. SDS-PAGE and Western blot analysis of cell lysates prepared
from NIH3T3/v-Src or EGF-stimulated NIH3T3/hEGFR was carried out using
antibodies against pShc, pErk1/2 (pp 42/pp 44), Erk, or β-actin, or
antiphosphotyrosine antibody, clone 4G10. Controls included nuclear
extracts untreated with compounds, and nuclear extracts or lysates
prepared from untreated cells.

[0313]Virtual high throughput screening relied upon computational modeling
of the native pTyr peptide sequence (APY*LKT; SEQ ID NO:1) from one Stat3
monomer bound within the SH2 domain of the second Stat3 monomer, as
observed for the Stat3 dimer protein bound to DNA (Becker, S. et al.
Nature, 1998, 394:145-151). For the docking studies, DNA was removed and
only one of the two monomers was employed. The approach was used to
evaluate the NCI Diversity Set and Plated Set chemical libraries.
Three-dimensional structures for compounds from the NCI Diversity Set and
NCI Plated Set were downloaded from NCI's DTP website (presented in
Siddiquee, K. et al. (2007)), processed with LigPrep (36; available from
Schrodinger, L. L. C.) to produce 2,392 3D structures for the Diversity
Set and 150,829 3D structures for the Plated Set. These structures were
docked using GLIDE 2.7 in SP (Standard Precision) mode into the pTyr
peptide binding site within the SH2 domain of the monomer employed. The
best scoring compounds (74 compounds selected via visual inspection from
the top 100 compounds with the best score from the Diversity Set and the
top 200 compounds from the Plated Set that then were filtered by MW
(<700) and computed logs (>-10) to yield 122 compounds) were
selected for evaluation in in vitro Stat3 DNA-binding assay. Nuclear
extracts containing activated STATs were incubated for 30 minutes with or
without increasing concentrations of compounds prior to incubation with
radiolabeled hSIE probe that binds to Stat1 and Stat3 or MGFe probe that
binds Stat1 and Stat5 and subjected to EMSA analysis. Results for a
representative number of compounds show differential inhibition of DNA
binding activity of Stat3 following preincubation of nuclear extracts
with compounds (Table 4) (data not shown for the remainder of the 194
compounds evaluated in the DNA-binding assay). Potent inhibition of Stat3
DNA-binding activity by the NCI Plated Set compounds, NSC 42067, NSC
59263, NSC 74859, and NSC 75912, was observed. Other notable potent
inhibitors of Stat3 DNA-binding activity from the Plated Set include NSC
11421, NSC 91529, and NSC 263435 (Table 4).

[0314]A few compounds with weak inhibitory activity were also identified
from the NCI Diversity Set (data not shown). To determine selectivity
against other STAT family members, selected active compounds were
evaluated in in vitro DNA-binding assay of the three STAT proteins,
Stat1, Stat3, and Stat5, in nuclear extracts prepared from EGF-stimulated
NIH3T3/hEGFR fibroblasts that activates all three STATs. EMSA analyses of
the DNA-binding activities of STAT proteins show that of the
representative compounds, NSC 74859 preferentially inhibits Stat3 over
Stat1 or Stat5 DNA-binding activity (Table 4), while NSC 42067 and NSC
59263 preferentially inhibit Stat3 and Stat5 over that of Stat1 (Table
4). In recognition of the role of constitutive Stat5 in hematological and
other cancers, the inhibition of Stat5 DNA-binding activity could have
clinical implications. Where Stat1 DNA-binding activity was inhibited, as
with NSC 42067 and NSC 59263, it occurred at concentrations 3-4 times
higher than concentrations that inhibited Stat3 or Stat5 (Table 4). In
contrast, NSC 75912 preferentially inhibits Stat1 over Stat3 or Stat5
(Table 4). For the remaining compounds, no appreciable pattern of
specificity of inhibition of the STAT family members was observed (Table
4 and data not shown). These studies identify NSC 42067, NSC 59263, and
NSC 74859 from the NCI chemical libraries as binders within the SH2
domain of Stat3 and which potently inhibit Stat3 DNA-binding activity.
The best of these compounds is NSC 74859 (re-synthesized as a pure sample
named BG2065p or S3I-201).

[0316]All patents, patent applications, provisional applications, and
publications referred to or cited herein, supra or infra, are
incorporated by reference in their entirety, including all figures and
tables, to the extent they are not inconsistent with the explicit
teachings of this specification.